Engineering Fc Antibody Regions to Confer Effector Function

ABSTRACT

The present invention relates to molecules having a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region. These modified molecules confer an effector function to a molecule, where the parent molecule does not detectably exhibit this effector function. In one embodiment, the variant Fc region binds FcγRIIIA and/or FcγRIIA with a greater affinity, relative to a comparable molecule comprising the wild-type Fc region. The molecules of the invention have particular utility in treatment, prevention or management of a disease or disorder, in a sub-population of patients, wherein the target antigen is expressed at low levels in the target cell population.

This application is a continuation of U.S. patent application Ser. No.12/565,911, filed Sep. 24, 2011, which application is a continuation ofU.S. patent application Ser. No. 11/271,140, filed Nov. 10, 2005 (nowissued as U.S. Pat. No. 7,632,497), which claims priority to U.S.Provisional Application Nos. 60/626,510 and 60/636,056, filed on Nov.10, 2004 and Dec. 13, 2004, respectively, all of which applications areincorporated herein by reference in their entireties and to whichpriority is claimed.

1. FIELD OF THE INVENTION

The present invention relates to molecules having a variant Fc region,wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region. These modified moleculesconfer an effector function to a molecule, where the parent moleculedoes not detectably exhibit this effector function. In particular, themolecules of the invention have an increased effector cell functionmediated by a FcγR, such as, but not limited to, ADCC. In oneembodiment, the variant Fc region binds FcγRIIIA and/or FcγRIIA with agreater affinity, relative to a comparable molecule comprising thewild-type Fc region. The molecules of the invention have particularutility in treatment, prevention or management of a disease or disorder,such as cancer, in a sub-population of patients, wherein the targetantigen is expressed at low levels in the target cell population, inparticular, in patients refractory to treatment with an existingtherapeutic antibody due to the low level of target antigen expressionon the cancer or associated cells.

2. BACKGROUND OF THE INVENTION 2.1 Fc Receptors and their Roles in theImmune System

The interaction of antibody-antigen complexes with cells of the immunesystem results in a wide array of responses, ranging from effectorfunctions such as antibody-dependent cytotoxicity, mast celldegranulation, and phagocytosis to immunomodulatory signals such asregulating lymphocyte proliferation and antibody secretion. All theseinteractions are initiated through the binding of the Fc domain ofantibodies or immune complexes to specialized cell surface receptors onhematopoietic cells. The diversity of cellular responses triggered byantibodies and immune complexes results from the structuralheterogeneity of Fc receptors. Fc receptors share structurally relatedligand binding domains which presumably mediate intracellular signaling.

The Fc receptors, members of the immunoglobulin gene superfamily ofproteins, are surface glycoproteins that can bind the Fc portion ofimmunoglobulin molecules. Each member of the family recognizesimmunoglobulins of one or more isotypes through a recognition domain onthe α chain of the Fc receptor. Fc receptors are defined by theirspecificity for immunoglobulin subtypes. Fc receptors for IgG arereferred to as FcγR, for IgE as FεR, and for IgA as FcαR. Differentaccessory cells bear Fc receptors for antibodies of different isotype,and the isotype of the antibody determines which accessory cells will beengaged in a given response (reviewed by Ravetch J. V. et al. 1991,Annu. Rev. Immunol. 9: 457-92; Gerber J. S. et al. 2001 Microbes andInfection, 3: 131-139; Billadeau D. D. et al. 2002, The Journal ofClinical Investigation, 2(109): 161-1681; Ravetch J. V. et al. 2000,Science, 290: 84-89; Ravetch J. V. et al., 2001 Annu. Rev. Immunol.19:275-90; Ravetch J. V. 1994, Cell, 78(4): 553-60). The different Fcreceptors, the cells that express them, and their isotype specificity issummarized in Table 1 (adapted from Immunobiology: The Immune System inHealth and Disease, 4^(th) ed. 1999, Elsevier Science Ltd/GarlandPublishing, New York).

Fcγ Receptors

Each member of this family is an integral membrane glycoprotein,possessing extracellular domains related to a C2-set ofimmunoglobulin-related domains, a single membrane spanning domain and anintracytoplasmic domain of variable length. There are three known FcγRs,designated FcγRI(CD64), FcγRII(CD32), and FcγRIII(CD16). The threereceptors are encoded by distinct genes; however, the extensive homologybetween the three family members suggest they arose from a commonprogenitor perhaps by gene duplication.

FcγRII (CD32)

FcγRII proteins are 40 KDa integral membrane glycoproteins which bindonly the complexed IgG due to a low affinity for monomeric Ig (10⁶ M⁻¹).This receptor is the most widely expressed FcγR, present on allhematopoietic cells, including monocytes, macrophages, B cells, NKcells, neutrophils, mast cells, and platelets. FcγRII has only twoimmunoglobulin-like regions in its immunoglobulin binding chain andhence a much lower affinity for IgG than FcγRI. There are three humanFcγRII genes (FcγRII-A, FcγRII-B, FcγRII-C), all of which bind IgG inaggregates or immune complexes.

Distinct differences within the cytoplasmic domains of FcγRII-A andFcγRII-B create two functionally heterogenous responses to receptorligation. The fundamental difference is that the A isoform initiatesintracellular signaling leading to cell activation such as phagocytosisand respiratory burst, whereas the β isoform initiates inhibitorysignals, e.g., inhibiting B-cell activation.

Signaling Through FcγRs

Both activating and inhibitory signals are transduced through the FcγRsfollowing ligation. These diametrically opposing functions result fromstructural differences among the different receptor isoforms. Twodistinct domains within the cytoplasmic signaling domains of thereceptor called immunoreceptor tyrosine based activation motifs (ITAMs)or immunoreceptor tyrosine based inhibitory motifs (ITIMS) account forthe different responses. The recruitment of different cytoplasmicenzymes to these structures dictates the outcome of the FcγR-mediatedcellular responses. ITAM-containing FcγR complexes include FcγRI,FcγRIIA, FcγRIIIA, whereas ITIM-containing complexes only includeFcγRIIB.

Human neutrophils express the FcγRIIA gene. FcγRIIA clustering viaimmune complexes or specific antibody cross-linking serves to aggregateITAMs along with receptor-associated kinases which facilitate ITAMphosphorylation. ITAM phosphorylation serves as a docking site for Sykkinase, activation of which results in activation of downstreamsubstrates (e.g., PI₃K). Cellular activation leads to release ofproinflammatory mediators.

The FcγRIIB gene is expressed on B lymphocytes; its extracellular domainis 96% identical to FcγRIIA and binds IgG complexes in anindistinguishable manner. The presence of an ITIM in the cytoplasmicdomain of FcγRIIB defines this inhibitory subclass of FcγR. Recently themolecular basis of this inhibition was established. When coligated alongwith an activating FcγR, the ITIM in FcγRIIB becomes phosphorylated andattracts the SH2 domain of the inosital polyphosphate 5′-phosphatase(SHIP), which hydrolyzes phosphoinositol messengers released as aconsequence of ITAM-containing FcγR-mediated tyrosine kinase activation,consequently preventing the influx of intracellular Ca⁺⁺. Thuscrosslinking of FcγRIIB dampens the activating response to FcγR ligationand inhibits cellular responsiveness. B cell activation, B cellproliferation and antibody secretion is thus aborted.

Current approaches to optimize the Fc region function (e.g.,antibody-dependent cell mediated cytotoxicity (ADCC), complementdependent cytotoxicity (CDC) activity) in therapeutic monoclonalantibodies and soluble polypeptides fused to Fc regions have focused ona limited number of single amino acid changes based on structuralanalysis and/or computer aided designs. Alternative approaches inengineering Fc regions have focused on the glycosylation of the Fcregion to optimize Fc region function.

TABLE 1 Receptors for the Fc Regions of Immunoglobulin Isotype ReceptorBinding Cell Type Effect of Ligation FcγRI (CD64) IgG1 10⁸ M⁻¹Macrophages Uptake Neutrophils Stimulation Eosinophils Activation ofrespiratory Dendritic cells burst Induction of killing FcγRII-A (CD32)IgG1 2 × 10⁶ M⁻¹ Macrophages Uptake Neutrophils Granule releaseEosinophils Dendritic cells Platelets Langerhan cells FcγRII-B2 IgG1 2 ×10⁶ M⁻¹ Macrophages Uptake (CD32) Neutrophils Inhibition of StimulationEosinophils FcγRII-B1 IgG1 2 × 10⁶ M⁻¹ B cells No uptake (CD32) Mastcells Inhibition of Stimulation FcγRIII (CD16) IgG1 5 × 10⁵ M⁻¹ NK cellsInduction of Killing Eosinophil Macrophages Neutrophils Mast Cells FcεRIIgE 1010 M⁻¹ Mast cells Secretion of granules Eosinophil Basophils FcαRI(CD89) IgA1, IgA2 10⁷ M⁻¹ Macrophages Uptake Neutrophils Induction ofkilling Eosinophils

3. SUMMARY OF THE INVENTION

The present invention is based, in part, on the inventors' discovery ofmethods for engineering the Fc region of an antibody to confer one ormore effector function activities to a parent antibody, which parentantibody does not exhibit the particular effector function activity at adetectable level when tested against a target cell. Such methods ofengineering include introducing one or more amino acid modifications(substitutions, deletions or insertions) in one or more portions of theFc region, which modifications introduce a detectable level of theeffector function activity in the modified antibody. In certainembodiments, the modifications alter the parent antibody's affinity forcertain FcγR receptors (e.g., activating FcγRs, inhibitory FcγRs) andone or more effector functions, such as antibody-dependent cell mediatedcytotoxicity (ADCC). In other embodiments, the modifications conferhomo-oligomerization activity to the parent Fc region such thatoligomerization of the modified antibody cross-links cell-surfaceantigens, resulting in apoptosis, negative-growth regulation or cellkilling.

The inventors have found that modification of an Fc region of a chimeric2B6 antibody (anti-FcγRIIB antibody) surprisingly conferred an effectorfunction activity (particularly, ADCC) on chimeric 2B6 antibodies, whichnormally exhibit no detectable ADCC in routine in vitro ADCC assays. Theinventors have found that modification of an Fc region of a chimeric 4D5antibody (anti-FcγRIIB antibody) surprisingly improved the effectorfunction activity (particularly, ADCC) of chimeric 4D5 antibodies incells with low levels of antigen expression. The inventors have furtherfound that modification of an Fc region of rituximab (anti-CD20monoclonal antibody) conferred effector function activity on therituximab antibody in cells from a patient population whose cells wereotherwise refractory to rituximab-induced effector function activity.

In one aspect, the invention encompasses molecules, preferablypolypeptides, and more preferably immunoglobulins (e.g., antibodies)comprising a variant Fc region having one or more amino acidmodifications (e.g., substitutions, but also including deletions orinsertions) in one or more Fc regions, relative to a parent molecule,which modifications confer a particular effector function activity onthe modified molecule, as compared to the parent molecule which haslittle or no detectable activity of that effector function (as measuredusing standard in vitro methods known in the art and exemplifiedherein). The effector function activities that may be conferred usingthe methods of the invention include, but are not limited to, ADCC,antibody-dependent phagocytosis, phagocytosis, opsonization,opsonophagocytosis, cell binding, rosetting, complement dependent cellmediated cytotoxicity (CDC).

Another aspect of the invention relates to molecules, preferablypolypeptides, and, more preferably, immunoglobulins (e.g., antibodies)comprising a variant Fc region having one or more amino acidmodifications (e.g., substitutions, deletions, insertions) in one ormore portions, which modifications increase the affinity and avidity ofthe variant Fc region for an FcγR (including activating and inhibitoryFcγRs). In some embodiments, said one or more amino acid modificationsincrease the affinity of the variant Fc region for FcγRIIIA and/orFcγRIIA. In another embodiment, the variant Fc region furtherspecifically binds FcγRIIB with a lower affinity than does the Fc regionof the comparable parent antibody (i.e., an antibody having the sameamino acid sequence as the antibody of the invention except for the oneor more amino acid modifications in the Fc region). In some embodiments,such modifications increase the affinity of the variant Fc region forFcγRIIIA and/or FcγRIIA and also enhance the affinity of the variant Fcregion for FcγRIIB relative to the parent antibody. In otherembodiments, said one or more amino acid modifications increase theaffinity of the variant Fc region for FcγRIIIA and/or FcγRIIA but do notalter the affinity of the variant Fc regions for FcγRIIB relative to theFc region of the parent antibody. In another embodiment, said one ormore amino acid modifications enhance the affinity of the variant Fcregion for FcγRIIIA and FcγRIIA but reduce the affinity for FcγRIIBrelative to the parent antibody.

Increased affinity and/or avidity results in detectable binding to theFcγR or detectable FcγR-related activity in cells that express lowlevels of the FcγR when binding activity of the parent molecule (withoutthe modified Fc region) cannot be detected on the cells. In certainembodiments the target antigen of the modified antibody is an FcγR, andthe modified antibody exhibits FcγR-binding or related activity in cellswhich express the target FcγR at a density of 10,000 molecules/cell orless, at a density of 5000 molecules/cell or less, at a density of 1000molecules/cell or less, at a density of 500 molecules or less, or at adensity of 200 molecules or less (but at least 10, at least 50, at least100 or at least 150 molecules/cell). In embodiments wherein the targetantigen is an FcγR, the increased binding to the on the cell surface maybe mediated by the CDR region of the antibody to an epitope on thetarget FcγR. Furthermore, this mechanism of increased antigen bindingmay occur with antibodies against non-Fcγ receptors or surface proteins.

The invention encompasses molecules, e.g., antibodies, with alteredaffinities and avidities for one or more target FcγRs. The antibodies ofthe invention with enhanced affinity and avidity for one or more targetFcγRs are particularly useful in cellular systems (for example forresearch or diagnostic purposes) where the FcγRs are expressed at lowlevels, for example, tumor specific B cells with low levels of FcγRIIB(e.g., non-Hodgkins lymphoma, CLL, and Burkitt's lymphoma). Although notintending to be bound by a particular mechanism of action, the moleculesof the invention with enhanced affinity and avidity for a particulartarget FcγR are valuable as research and diagnostic tools by enhancingthe sensitivity of detection of FcγRs which are normally undetectabledue to a low level of expression. The antibodies of the invention withenhanced affinity and avidity for FcγRs are particularly useful for thetreatment, prevention or management of a cancer, or another disease ordisorder, in a subject, wherein the FcγRs are expressed at low levels inthe target cell populations. As used herein, FcγR expression in cells isdefined in terms of the density of such molecules per cell as measuredusing common methods known to those skilled in the art. The molecules ofthe invention comprising variant Fc regions preferably also have anenhanced avidity and affinity and/or effector function in cells whichexpress a target antigen to which the modified antibodyimmunospecifically binds, e.g., a cancer antigen, at low density, forexample, at a density of 30,000 to 20,000 molecules/cell, at a densityof 20,000 to 10,000 molecules/cell, at a density of 10,000 to 5,000molecules/cell, at a density of 5,000 to 1,000 molecules/cell, at adensity of 1,000 to 200 molecules/cell or at a density of 200molecules/cell or less. The molecules of the invention have particularutility in treatment, prevention or management of a disease or disorder,such as cancer, in a sub-population of patients, wherein the targetantigen is expressed at low levels in the target cell population, inparticular, in patients refractory to treatment with an existingtherapeutic antibody due to the low level of target antigen expressionon the cancer or other cells associated with the disease or disorder tobe treated, prevented or managed.

The invention encompasses engineering human, chimeric or humanizedtherapeutic antibodies in the Fc region by modifying one or more Fcregion amino acids, which modifications alter the detectable affinityand avidity of the antibodies for one or more target antigens, e.g.,FcγR receptors or cancer antigens, and/or the detectable effectorfunction activity or cell killing activity of the modified antibody. Inone embodiment, said one or more modifications to the amino acids of theFc region enhance the affinity and avidity of the antibody for one ormore target antigens, e.g., FcγR receptors or cancer antigens. Thesetherapeutic antibodies, by virtue of the modifications of the invention,have increased efficacy in patients refractory to treatment with theparent antibody, due, in certain instances, to reduced levels of theexpression of the target antigen, as well as in patients who respond tothe parent antibody.

Although not intending to be bound by a particular mechanism of action,therapeutic antibodies engineered in accordance with the invention haveenhanced therapeutic efficacy, in part, due to the ability of the Fcportion of the antibody to bind a target cell which expresses theparticular FcγRs at reduced levels, for example, by virtue of theability of the antibody to remain on the target cell longer due to animproved off rate for Fc-FcγR interaction. In another embodiment, saidone or more modifications to the amino acids of the Fc region modifiesthe affinity and avidity of the antibody for one or more FcγR receptors.In a specific embodiment, the invention encompasses antibodiescomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to the parent Fc region,which variant Fc region only binds one FcγR, wherein said FcγR isFcγRIIIA. In another specific embodiment, the invention encompassesantibodies comprising a variant Fc region, wherein said variant Fcregion comprises at least one amino acid modification relative to theparent Fc region, which variant Fc region only binds one FcγR, whereinsaid FcγR is FcγRIIA. In yet another embodiment, the inventionencompasses antibodies comprising a variant Fc region, wherein saidvariant Fc region comprises at least one amino acid modificationrelative to the parent Fc region, which variant Fc region only binds oneFcγR, wherein said FcγR is FcγRIIB.

The affinities and binding properties of the antibodies of the inventionfor the target antigen or an FcγR are initially determined using invitro assays (biochemical or immunological based assays) known in theart for determining antigen-antibody or Fc-FcγR interactions (i.e.,specific binding of an Fc region to an FcγR), respectively, includingbut not limited to, ELISA assay, surface plasmon resonance assay orimmunoprecipitation assay. Preferably, the binding properties of themolecules of the invention are also characterized by in vitro functionalassays for determining one or more FcγR mediated effector cellfunctions. In most preferred embodiments, the antibodies of theinvention have similar binding properties in in vivo models (such asthose described and disclosed herein) as those in in vitro based assays.However, the present invention does not exclude molecules of theinvention that do not exhibit the desired phenotype in in vitro basedassays but do exhibit the desired phenotype in vivo.

The invention also encompasses molecules, preferably polypeptides, andmore preferably, immunoglobulins (e.g., antibodies) comprising a variantFc region having one or more amino acid modifications (e.g.,substitutions, deletions, insertions) in one or more portions, whichmodifications confer detectable effector function activity to themolecule not detectable in the parent molecule. In certain embodiments,the parent molecule is an antibody. In yet other embodiments, the parentantibody is rituximab or humanized 2B6 (see U.S. Patent ApplicationPublication 2004/0185045 and U.S. patent application Ser. No.11/126,978, filed May 10, 2005, by Johnson et al., which areincorporated herein by reference in their entireties), and the modifiedantibodies are used to treat the indications associated with the parentantibodies. Although not intending to be bound to a particular mechanismof action, the molecules of the invention with conferred effectorfunction activity are particularly useful for the treatment and/orprevention of a disease or disorder where an effector cell function(e.g., ADCC) mediated by an FcγR is desired (e.g., cancer, infectiousdisease). Alternately, the molecules of the invention with Fcmodifications may exhibit enhanced therapeutic efficacy due to theintroduction of homo-oligomerization activity in the Fc region,resulting in apoptosis, negative-growth regulation or cell killingassociated with surface antigen cross-linking.

The invention encompasses methods and compositions for treatment,prevention or management of a cancer in a subject, comprisingadministering to the subject a therapeutically effective amount of oneor more molecules comprising a variant Fc region engineered inaccordance with the invention, which molecule further binds a cancerantigen. In certain embodiments, the subject is human. In otherembodiments, the molecules of the invention are modified rituximab, andare preferably used in the treatment of lymphoma, such as Non-Hodgkinslymphoma, or modified humanized 2B6 antibodies engineered according tothe methods of the invention, which modified antibodies possess the sameindications as the parent antibodies. Molecules of the inventioncomprising the variant Fc regions are particularly useful for theprevention, inhibition, reduction of growth or regression of primarytumors, or metastasis of cancer cells. Although not intending to bebound by a particular mechanism of action, molecules of the inventionenhance the efficacy of cancer therapeutics by i) enhancing antibodymediated effector function or ii) enhancing the apoptosis signaling,negative-growth regulation or cell killing associated with surfaceantigen cross-linking by introducing homo-oligomerization activity inthe modified molecules, resulting in an enhanced rate of tumor clearanceor an enhanced rated of tumor reduction or a combination thereof.

According to an aspect of the invention, immunotherapeutics may bemodifying in accordance with the invention to increase the potency of anantibody effector function activity, e.g., ADCC, CDC, phagocytosis,opsonization, etc. In a specific embodiment, antibody dependent cellulartoxicity and/or phagocytosis (e.g., of tumor cells) is enhanced bymodifying immunotherapeutics with variant Fc regions of the invention.Molecules of the invention may render immunotherapy cancer treatmentefficacious in a patient population by enhancing (or renderingdetectable) at least one antibody-mediated effector function activity.In one particular embodiment, the efficacy of immunotherapy treatment isenhanced by rendering the complement dependent cascade activitydetectable. In another embodiment of the invention, the efficacy ofimmunotherapy treatment is enhanced by rendering the phagocytosis and/oropsonization of the targeted cells, e.g., tumor cells, detectable. Inanother embodiment of the invention, the efficacy of treatment isenhanced by enhancing antibody-dependent cell-mediated cytotoxicity(“ADCC”) in destruction of the targeted cells, e.g., tumor cells,detectable. Determining whether such activity is detectable is doneusing routine assays known in the art and described herein.

In a specific embodiment, the invention encompasses a moleculecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to the parent Fc regionsuch that the molecule has an enhanced effector activity, provided saidone or more amino acid modifications includes substitutions at one ormore positions. The amino acid positions recited herein are numberedaccording to the EU index as set forth in Kabat et al., Sequence ofProteins of Immunological Interest, 5^(th) Ed. Public Health Service,NH1, MD (1991), expressly incorporated herein by reference. In aspecific embodiment, the variant Fc region has a leucine at position247, a lysine at position 421, or a glutamic acid at position 270. Inother specific embodiments, the variant Fc region has a leucine atposition 247, a lysine at position 421 and a glutamic acid at position270 (MgFc31/60); a threonine at position 392, a leucine at position 396,a glutamic acid at position 270, and a leucine at position 243(MgFc38/60/F243L); a histidine at position 419, a leucine at position396, and a glutamic acid at position 270 (MGFc51/60); an alanine atposition 240, a leucine at position 396, and a glutamic acid at position270 (MGFc52/60); a histidine at position 419, a leucine at position 396,a glutamic acid at position 270, and a leucine at position 243(MGFc51/60/F243L); a lysine at position 255 and a leucine at position396 (MgFc55); a lysine at position 255, a leucine at position 396, and aglutamic acid at position 270 (MGFc55/60); a lysine at position 255, aleucine at position 396, a glutamic acid at position 270, and a lysineat position 300 (MGFc55/60/Y300L); a lysine at position 255, a leucineat position 396, a glutamic acid at position 270, and a leucine atposition 243 (MgFc55/60/F243L); a lysine at position 255, a leucine atposition 396, a glutamic acid at position 270, and a glycine at position292 (MgFc55/60/R292G); a glutamic acid at position 370, a leucine atposition 396, and a glutamic acid at position 270 (MGFc59/60); aglutamic acid at position 270, an aspartic acid at position 316, and aglycine at position 416 (MgFc71); a leucine at position 243, a prolineat position 292, an isoleucine at position 305, and a leucine atposition 396 (MGFc74/P396L); a leucine at position 243, a glutamic acidat position 270, a glycine at position 292, and a leucine at position396; a leucine at position 243, a lysine at position 255, a glutamicacid at position 270, and a leucine at position 396; or a glutamine atposition 297.

The invention also encompasses methods for treating or preventing aninfectious disease in a subject comprising administering atherapeutically or prophylactically effective amount of one or moremolecules of the invention that bind an infectious agent or cellularreceptor therefore. Infectious diseases that can be treated or preventedby the molecules of the invention are caused by infectious agentsincluding but not limited to viruses, bacteria, fungi, protozae, andviruses. Although not intending to be bound by a particular mechanism ofaction, the methods and/or molecules of the invention confer atherapeutic effect not detectable in the parent antibody or enhance thetherapeutic effect of the parent antibody by i) enhancing or renderingdetectable the antibody mediated effector function toward an infectiousagent or ii) enhancing or rendering detectable the apoptosis signaling,negative-growth regulation or cell killing associated with surfaceantigen cross-linking by conferring homo-oligomerization activity in themodified molecules.

According to one aspect of the invention, molecules of the inventioncomprising variant Fc regions have detectable antibody effector functiontowards an infectious agent, which was not detectable in the parentmolecule comprising a wild-type Fc region. In a specific embodiment,molecules of the invention enhance the efficacy of treatment of aninfectious disease by enhancing or rendering detectable phagocytosisand/or opsonization of the infectious agent causing the infectiousdisease. In another specific embodiment, molecules of the inventionenhance the efficacy of treatment of an infectious disease by enhancingor rendering detectable ADCC of infected cells causing the infectiousdisease.

The invention encompasses characterization of the molecules of theinvention (e.g., therapeutic monoclonal antibodies engineered accordingto the methods of the invention) using assays known to those skilled inthe art for identifying the effector cell function of the molecules. Inparticular, the invention encompasses characterizing the molecules ofthe invention for FcγR-mediated effector cell function. Examples ofeffector cell functions that can be assayed in accordance with theinvention, include but are not limited to, antibody-dependent cellmediated cytotoxicity (ADCC), phagocytosis, opsonization,opsonophagocytosis, C1q binding, and complement dependent cell mediatedcytotoxicity (CDC). Cell-based or cell free assays for determiningeffector cell function activity are routine and known to those skilledin the art and described herein.

In one embodiment, the molecules of the invention can be assayed forFcγR-mediated phagocytosis in human monocytes. Alternatively, theFcγR-mediated phagocytosis of the molecules of the invention may beassayed in other phagocytes, e.g., neutrophils (polymorphonuclearleuckocytes; PMN); human peripheral blood monocytes, monocyte-derivedmacrophages, which can be obtained using standard procedures known tothose skilled in the art. In another embodiment, the molecules of theinvention may be assayed using an antibody-dependent opsonophagocytosisassay (ADCP). In yet another embodiment, the molecules of the inventioncan be assayed for FcγR-mediated ADCC activity in effector cells, e.g.,natural killer cells, using any of the standard methods known to thoseskilled in the art. In yet another embodiment, the molecules of theinvention are characterized for antibody dependent cellular cytotoxicity(ADCC).

Preferably, the effector cells used in the ADCC assays of the inventionare peripheral blood mononuclear cells (PBMC) that are purified fromnormal human blood, using standard methods known to one skilled in theart, e.g., using Ficoll-Paque density gradient centrifugation. Preferredeffector cells for use in the methods of the invention express differentFcγR activating receptors. The invention encompasses, effector cellsexpressing FcγRI, FcγRIIA and FcγRIIB, and monocyte derived primarymacrophages derived from whole human blood expressing both FcγRIIIA andFcγRIIB. Both the ratio of effector cell:target cell and concentrationof antibody to be used in the functional assays in accordance with theinvention will be appreciated to be dependent of the particular assayand system to be tested. The invention encompasses use of the effectorcells in the functional assays of effector function activity at aneffector cell:target cell ratio of 1:1, 10:1, 30:1, 60:1, 75:1 or 100:1.The invention encompasses the use of antibody in the functional assaysof effector function activity at an concentration of 0.2 μg/ml to 3μg/ml, 0.5 μg/ml to 2 μg/ml or 0.5 μg/ml to 1 μg/ml.

In another embodiment, the molecules of the invention may be assayed forC1q binding, which mediates complement dependent cytotoxicity (CDC). Todetermine C1q binding, a C1q binding ELISA may be performed. To assesscomplement activation, a complement dependent cytotoxicity (CDC) assaymay be performed using standard methods known in the art.

The Fc variants of the present invention may be combined with other Fcmodifications known in the art. The invention encompasses combining anFc variant of the invention with other Fc modifications to provideadditive, synergistic, or novel properties to the modified antibody.Preferably, the Fc variants of the invention enhance the phenotype ofthe modification with which they are combined. For example, if an Fcvariant of the invention is combined with a mutant known to bindFcγRIIIA with a higher affinity than a comparable wild type Fc region;the combination with a mutant of the invention results in a greater foldenhancement in FcγRIIIA affinity.

In one embodiment, the Fc variants of the present invention may becombined with other known Fc variants such as those disclosed in Duncanet al, 1988, Nature 332:563-564; Lund et al., 1991, J. Immunol.147:2657-2662; Lund et al, 1992, Mol Immunol 29:53-59; Alegre et al,1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl.Acad Sci USA 92:11980-11984; Jefferis et al, 1995, Immunol Lett.44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et al, 1996,Immunol Lett 54:101-104; Lund et al, 1996, J Immunol 157:49634969;Armour et al, 1999, Eur J Immunol 29:2613-2624; Idusogie et al, 2000, JImmunol 164:41784184; Reddy et al, 2000, J Immunol 164:1925-1933; Xu etal., 2000, Cell Immunol 200:16-26; Idusogie et al, 2001, J Immunol166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferiset al, 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem SocTrans 30:487-490); U.S. Pat. No. 5,624,821; U.S. Pat. No. 5,885,573;U.S. Pat. No. 6,194,551; PCT WO 00/42072; PCT WO 99/58572; each of whichis incorporated herein by reference in its entirety.

The present invention also encompasses antibodies that are homodimers orheterodimers of Fc regions. Homodimeric or heterodimeric antibodies ofthe invention comprise variant Fc regions, wherein the two Fc chainshave the same or different amino acid sequences, respectively. In oneembodiment, each Fc chain of the heterodimeric antibody comprises one ormore different amino acid modifications relative to the other chain. Inanother embodiment, one Fc chain of the heterodimeric antibody comprisesthe wild type Fc chain and the other Fc chain comprises one or moreamino acid modifications relative to the wild type chain.

The present invention also includes polynucleotides that encode amolecule of the invention, including polypeptides and antibodies,identified by the methods of the invention. The polynucleotides encodingthe molecules of the invention may be obtained, and the nucleotidesequence of the polynucleotides determined, by any method known in theart. The invention relates to an isolated nucleic acid encoding amolecule of the invention. The invention also provides a vectorcomprising said nucleic acid. The invention further provides host cellscontaining the vectors or polynucleotides of the invention.

The invention further provides methods for the production of themolecules of the invention. The molecules of the invention, includingpolypeptides and antibodies, can be produced by any method known tothose skilled in the art, in particular, by recombinant techniques. Incertain embodiments, antibodies of the invention are created byengineering mutations identified as conferring therapeuticallyeffective, detectable, effector function activity into the Fc regions ofantibodies which do not natively exhibit such activity. In otherembodiments, the invention relates to a method for recombinantlyproducing a molecule of the invention, said method comprising: (i)culturing in a medium a host cell comprising a nucleic acid encodingsaid molecule, under conditions suitable for the expression of saidmolecule; and (ii) recovery of said molecule from said medium.

The invention also encompasses methods for improving the therapeuticefficacy of molecules, preferably polypeptides, and more preferablyimmunoglobulins (e.g., antibodies) comprising a variant Fc regionhaving, which Fc regions have been engineered according to the methodsof the invention, which engineering confers detectable effector functionactivity on the modified molecule, as compared to the parent moleculewhich exhibited little or no detectable activity of that effectorfunction (as measured using standard in vitro methods known in the artand exemplified herein).

The invention provides pharmaceutical compositions comprising a moleculeof the invention, e.g., a polypeptide comprising a variant Fc region, animmunoglobulin comprising a variant Fc region, a therapeutic antibodyengineered in accordance with the invention, and a pharmaceuticallyacceptable carrier. The invention additionally provides pharmaceuticalcompositions further comprising one or more additional therapeuticagents, including but not limited to anti-cancer agents,anti-inflammatory agents, immunomodulatory agents.

In detail, the invention thus pertains to a modified antibody that bindsan antigen, the modified antibody comprising a variant human IgG Fcregion, wherein the variant human IgG Fc region comprises at least oneamino acid modification relative to the human IgG Fc region of a parentantibody that binds the antigen, such that the modified antibodyexhibits, in an in vitro assay, detectable effector function activity(especially phagocytosis, opsonization, cell binding, rosetting,complement dependent cell mediated cytotoxicity (CDC), or antibodydependent cell-mediated cell cytotoxicity (ADCC)) in cells positive forthe antigen, wherein the parent antibody does not exhibit detectableeffector function activity in the cells using the in vitro assay (andespecially wherein the in vitro assay is performed at an effectorcell:target cell ratio of 10:1, 30:1, 50:1, 75:1 or 100:1). Theinvention particularly pertains to all such modified antibodies, whereinthe human IgG Fc region is a human IgG1, IgG2, IgG3, or IgG4 Fc region.

The invention further pertains to a modified antibody that binds anantigen, the modified antibody comprising a variant human IgG Fc region,wherein the variant human IgG Fc region comprises at least one aminoacid modification relative to the human IgG Fc region of a parentantibody that binds the antigen, such that the modified antibody istherapeutically effective in a patient refractory to treatment with theparent antibody. The invention particularly pertains to such a modifiedantibodies, wherein the modified antibody exhibits, in an in vitroassay, detectable effector function activity (especially phagocytosis,opsonization, cell binding, rosetting, complement dependent cellmediated cytotoxicity (CDC), or antibody dependent cell-mediated cellcytotoxicity (ADCC)) in cells derived from the patient, which cells arepositive for the antigen, wherein the parent antibody does not exhibitdetectable function activity in the cells using the in vitro assay (andespecially wherein the in vitro assay is performed at an effectorcell:target cell ratio of 10:1, 30:1, 50:1, 75:1 or 100:1). Theinvention particularly pertains to all such modified antibodies, whereinthe human IgG Fc region is a human IgG1, IgG2, IgG3, or IgG4 Fc region.

The invention particularly pertains to all such modified antibodies,wherein the antigen is a cancer antigen (for example, MAGE-1, MAGE-3,BAGE, GAGE-1, GAGE-2, N-acetylglucosaminyltransferase, p15,beta-catenin, MUM-1, CDK4, HER-2/neu, human papillomavirus-E6, humanpapillomavirus-E7, MUC-1, CD20 or CD32B) or an antigen that is expressedon the surface of a cell.

The invention particularly pertains to all such modified antibodies,wherein the parent antibody is HERCEPTIN®, IC14, PANOREX™, IMC-225,VITAXIN™, CAMPATH™ 1H/LDP-03, LYMPHOCIDE™, ZEVLIN™ or rituximab.

The invention particularly pertains to all such modified antibodies,wherein the at least one amino acid modification comprises substitutionat position 370 with glutamic acid, at position 396 with leucine and atposition 270 with glutamic acid; at position 419 with histidine, atposition 396 with leucine and at position 270 with glutamic acid; atposition 240 with alanine, at position 396 with leucine and at position270 with glutamic acid; at position 240 with alanine, at position 396with leucine and at position 270 with glutamic acid; at position 255with leucine, at position 396 with leucine and position 270 withglutamic acid; at position 255 with leucine, at position 396 withleucine, at position 270 with glutamic acid and at position 292 glycine;at position 255 with leucine, at position 396 with leucine, at position270 with glutamic acid and at position 300 leucine; at position 243 withleucine, at position 270 with glutamic acid, at position 392 withasparagine and at position 396 with leucine; or at position 243 withleucine, at position 255 with leucine, at position 270 with glutamicacid and at position 396 with leucine.

The invention particularly pertains to all such modified antibodies,wherein the amino acid modification comprises at least one amino acidmodification in the CH2 domain (and especially wherein the amino acidmodification in the CH2 domain comprises substitution at position 240,243, 247, 255, 270, 292, or 300 with another amino acid at thatposition), and/or in wherein the amino acid modification comprises atleast one amino acid modification in the CH3 domain (and especiallywherein the amino acid modification in the CH3 domain comprisessubstitution at position 370, 392, 396, 419, or 421 with another aminoacid at that position) and/or in the hinge region of the human IgG heavychain.

The invention particularly pertains to all such modified antibodies,wherein the variant IgG Fc region specifically binds FcγRIIIA with agreater affinity than the parent antibody binds FcγRIIIA and especiallywherein the variant IgG Fc region specifically binds FcγRIIB with alower affinity than the parent antibody binds FcγRIIB

The invention particularly pertains to all such modified antibodies,wherein the variant IgG Fc region specifically binds FcγRIIA with agreater affinity than the parent antibody binds FcγRIIA and especiallywherein the variant IgG Fc region specifically binds FcγRIIB with alower affinity than the parent antibody binds FcγRIIB

The invention particularly pertains to all such modified antibodies,wherein the variant IgG Fc region specifically binds FcγRIIB with alower affinity than the parent antibody binds FcγRIIB.

The invention particularly pertains to all such modified antibodies,which detectably binds cells positive for the antigen, which antigen isexpressed at a density of 200 to 1,000 molecules/cell on the cells.

The invention particularly pertains to all such modified antibodies,wherein the antibody is a monoclonal antibody or a chimeric, human orhumanized antibody.

The invention particularly pertains to all such modified antibodies,wherein the antibody is a chimeric 2B6 antibody.

The invention additionally pertains to such modified antibodies, whereinthe antigen is associated with an infectious disease (an especially inwhich the antigen is a viral, bacterial or fungal antigen).

The invention additionally pertains to such modified antibodies, whereinthe parent antibody has immunomodulatory activity.

The invention additionally pertains to a method of treating cancer in apatient (especially a human patient) having a cancer characterized by acancer antigen (especially MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2,N-acetylglucosaminyltransferase, p15, beta-catenin, MUM-1, CDK4,HER-2/neu, human papillomavirus-E6, human papillomavirus-E7, MUC-1, CD20or CD32B), wherein the method comprises administering to the patient atherapeutically effective amount of the above-described modifiedantibody that binds a cancer antigen (and especially in the embodimentin which the patient is refractory to treatment with the parent antibody(especially wherein the parent antibody is HERCEPTIN®, IC14, PANOREX™,IMC-225, VITAXIN™, Campath 1H/LDP-03, LYMPHOCIDE™, ZEVLIN™ orrituximab). The invention additionally pertains to the use of such amethod of treating cancer that further comprises the administration ofone or more additional cancer therapies.

The invention additionally pertains to such a method of treating cancerin a patient, wherein the modified antibody binds a cancer antigen thatis a colon, breast, ovarian, prostate, cervical, pancreatic carcinoma,non-Hodgkins lymphoma or chronic lymphocytic leukemia antigen.

The invention particularly pertains to all such methods of treatmentwherein the modified antibody is a monoclonal antibody or a chimeric,human or humanized antibody.

The invention particularly pertains to all such methods of treatmentwherein the human IgG Fc region is a human IgG1, IgG2, IgG3, or IgG4 Fcregion.

The invention further pertains to a pharmaceutical compositioncomprising any of the above-described modified antibodies and apharmaceutically acceptable carrier.

The invention further pertains to a nucleotide sequence encoding a heavyor a light chain of any of the above-described modified antibodies, andespecially a vector containing such a sequence. The invention furtherpertains to a host cell comprising such nucleic acid molecule or suchvector. In particular, the invention concerns a host cell comprising afirst nucleotide sequence encoding a heavy chain of a modified antibodyand a second nucleotide sequence encoding a light chain of a modifiedantibody, the modified antibody being any of those described above.

The invention further pertains to a method for recombinantly producing amodified antibody, the method comprising:

-   -   a. culturing in a medium the host cell of claim 46 under        conditions suitable for the expression of the modified antibody;        and    -   b. recovery of the modified antibody from the medium.

The invention further pertains to a method for improving a therapeuticantibody that specifically binds to an antigen and that does notexhibit, in an in vitro assay, detectable effector function activity,the method comprising

-   -   a. introducing at least one amino acid modification in the Fc        region of the therapeutic antibody to generate a modified        therapeutic antibody; and    -   b. determining whether the modified therapeutic antibody        exhibits detectable effector function activity using the in        vitro assay.

The invention particularly pertains to such a method for improving atherapeutic antibody, wherein the modified antibody exhibits any set orsubset of the following attributes: ability to bind to cells expressingthe antigen at a density of 200 to 1,000 molecules/cell, ability to bindto a FcγRIIA or FcγRIIIA target molecule, ability to bind to an antigentarget molecule, ability to bind to a cancer antigen target molecule,and exhibiting an increase in at least one effector function.

The invention further pertains to a modified antibody that binds anantigen, the modified antibody comprising a variant human IgG Fc region,wherein the variant human IgG Fc region comprises at least one aminoacid modification relative to the human IgG Fc region of a parentantibody that binds the antigen, such that the modified antibodyexhibits, in an in vitro assay, detectable cell killing in cellspositive for the antigen, wherein the parent antibody does not exhibitdetectable cell killing in the cells using the in vitro assay.

The invention further pertains to a method of treating cancer in apatient having a cancer characterized by a cancer antigen, the methodcomprising administering to the patient a therapeutically effectiveamount of the modified antibody of claim 54, which modified antibodybinds the cancer antigen.

The invention further pertains to a modified antibody that binds anantigen, the modified antibody comprising a variant human IgG Fc region,wherein the variant human IgG Fc region comprises at least one aminoacid modification relative to the human IgG Fc region of a parentantibody that binds the antigen, such that the modified antibodyexhibits, in an in vitro assay, detectable effector function activity incells positive for the antigen, wherein the parent antibody does notexhibit detectable effector function activity in the cells using the invitro assay, which in vitro assay is performed at an effectorcell:target cell ratio of 75:1, which in vitro assay is performed at aneffector cell:target cell ratio of 30:1 or which in vitro assay isperformed at an effector cell:target cell ratio of 10:1.

The invention further pertains to a modified antibody that binds anantigen, the modified antibody comprising a variant human IgG Fc region,wherein the variant human IgG Fc region comprises at least one aminoacid modification relative to the human IgG Fc region of a parentantibody that binds the antigen, such that the modified antibodyexhibits, in an in vitro assay, detectable cell killing in cellspositive for the antigen, wherein the parent antibody does not exhibitdetectable cell killing in the cells using the in vitro assay, which invitro assay is performed at an effector cell:target cell ratio of 75:1,which in vitro assay is performed at an effector cell:target cell ratioof 30:1, or which in vitro assay is performed at an effector cell:targetcell ratio of 10:1.

3.1 Definitions

As used herein, the term “Fc region” is used to define a C-terminalregion of an IgG heavy chain. Throughout the present specification, thenumbering of the residues in an IgG heavy chain is that of the EU indexas in Kabat et al., Sequences of Proteins of Immunological Interest,5^(th) Ed. Public Health Service, NH1, MD (1991), expressly incorporatedherein by references. The “EU index as in Kabat” refers to the numberingof the human IgG1 EU antibody. An example of the amino acid sequencecontaining the human IgG1 Fc region is SEQ ID NO:11 and is shown inFIG. 1. SEQ ID NO:11 and FIG. 1 set forth the amino acid sequence of theIgG1 hinge-Fc region. Although boundaries may vary slightly, as numberedaccording to the Kabat system, the Fc domain extends from amino acid 231to amino acid 447 (which corresponds to amino acid 16 to amino acid 232as numbered in SEQ ID NO:11 and FIG. 1).

The Fc region of an IgG comprises two constant domains, CH2 and CH3. TheCH2 domain of a human IgG Fc region usually extends from amino acids 231to amino acid 341 according to the numbering system of Kabat(corresponding to amino acids 16 to 126 as numbered in SEQ ID NO:11 andFIG. 1). The CH3 domain of a human IgG Fc region usually extends fromamino acids 342 to 447 according to the numbering system of Kabat(corresponding to amino acids 127 to 232 as numbered in SEQ ID NO:11 andFIG. 1). The CH2 domain of a human IgG Fc region (also referred to as“Cγ2” domain) is unique in that it is not closely paired with anotherdomain. Rather, two N-linked branched carbohydrate chains are interposedbetween the two CH2 domains of an intact native IgG.

The “hinge region” is generally defined as stretching from Glu216 toPro230 of human IgG1 (corresponding to amino acids 1-15 as numbered inSEQ ID NO:11 and FIG. 1). Hinge regions of other IgG isotypes may bealigned with the IgG1 sequence by placing the first and last cysteineresidues forming inter-heavy chain S—S binds in the same positions.

As used herein, the terms “antibody” and “antibodies” refer tomonoclonal antibodies, multispecific antibodies, human antibodies,humanized antibodies, synthetic antibodies, chimeric antibodies,polyclonal antibodies, camelized antibodies, single-chain Fvs (scFv),single chain antibodies, Fab fragments, F(ab′) fragments,disulfide-linked bispecific Fvs (sdFv), intrabodies, and anti-idiotypic(anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Idantibodies to antibodies of the invention), and epitope-bindingfragments of any of the above. In particular, antibodies includeimmunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site. Immunoglobulin molecules can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁and IgA₂) or subclass.

As used herein, the term “derivative” in the context of polypeptides orproteins refers to a polypeptide or protein that comprises an amino acidsequence which has been altered by the introduction of amino acidresidue substitutions, deletions or additions. The term “derivative” asused herein also refers to a polypeptide or protein which has beenmodified, i.e, by the covalent attachment of any type of molecule to thepolypeptide or protein. For example, but not by way of limitation, anantibody may be modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. A derivative polypeptide or protein may beproduced by chemical modifications using techniques known to those ofskill in the art, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Further, a derivative polypeptide or protein derivative possesses asimilar or identical function as the polypeptide or protein from whichit was derived.

As used herein, the term “derivative” in the context of anon-proteinaceous derivative refers to a second organic or inorganicmolecule that is formed based upon the structure of a first organic orinorganic molecule. A derivative of an organic molecule includes, but isnot limited to, a molecule modified, e.g., by the addition or deletionof a hydroxyl, methyl, ethyl, carboxyl or amine group. An organicmolecule may also be esterified, alkylated and/or phosphorylated.

As used herein, the terms “disorder” and “disease” are usedinterchangeably to refer to a condition in a subject. In particular, theterm “autoimmune disease” is used interchangeably with the term“autoimmune disorder” to refer to a condition in a subject characterizedby cellular, tissue and/or organ injury caused by an immunologicreaction of the subject to its own cells, tissues and/or organs. Theterm “inflammatory disease” is used interchangeably with the term“inflammatory disorder” to refer to a condition in a subjectcharacterized by inflammation, preferably chronic inflammation.Autoimmune disorders may or may not be associated with inflammation.Moreover, inflammation may or may not be caused by an autoimmunedisorder. Thus, certain disorders may be characterized as bothautoimmune and inflammatory disorders.

As used herein, the term “cancer” refers to a neoplasm or tumorresulting from abnormal uncontrolled growth of cells. As used herein,cancer explicitly includes leukemias and lymphomas. In some embodiments,cancer refers to a benign tumor, which has remained localized. In otherembodiments, cancer refers to a malignant tumor, which has invaded anddestroyed neighboring body structures and spread to distant sites. Insome embodiments, the cancer is associated with a specific cancerantigen that is expressed on cancer cells.

As used herein, the term “immunomodulatory agent” and variations thereofrefer to an agent that modulates a host's immune system. In certainembodiments, an immunomodulatory agent is an immunosuppressant agent. Incertain other embodiments, an immunomodulatory agent is animmunostimulatory agent. Immunomodatory agents include, but are notlimited to, small molecules, peptides, polypeptides, fusion proteins,antibodies, inorganic molecules, mimetic agents, and organic molecules.

As used herein, the term “epitope” refers to a fragment of a polypeptideor protein or a non-protein molecule having antigenic or immunogenicactivity in an animal, preferably in a mammal, and most preferably in ahuman. An epitope having immunogenic activity is a fragment of apolypeptide or protein that elicits an antibody response in an animal.An epitope having antigenic activity is a fragment of a polypeptide orprotein to which an antibody immunospecifically binds as determined byany method well-known to one of skill in the art, for example byimmunoassays. Antigenic epitopes need not necessarily be immunogenic.

As used herein, the term “fragment” refers to a peptide or polypeptidecomprising an amino acid sequence of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least contiguous 80 amino acid residues, atleast contiguous 90 amino acid residues, at least contiguous 100 aminoacid residues, at least contiguous 125 amino acid residues, at least 150contiguous amino acid residues, at least contiguous 175 amino acidresidues, at least contiguous 200 amino acid residues, or at leastcontiguous 250 amino acid residues of the amino acid sequence of anotherpolypeptide. In a specific embodiment, a fragment of a polypeptideretains at least one function of the polypeptide.

As used herein, the terms “nucleic acids” and “nucleotide sequences”include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g.,mRNA), combinations of DNA and RNA molecules or hybrid DNA/RNAmolecules, and analogs of DNA or RNA molecules. Such analogs can begenerated using, for example, nucleotide analogs, which include, but arenot limited to, inosine or tritylated bases. Such analogs can alsocomprise DNA or RNA molecules comprising modified backbones that lendbeneficial attributes to the molecules such as, for example, nucleaseresistance or an increased ability to cross cellular membranes. Thenucleic acids or nucleotide sequences can be single-stranded,double-stranded, may contain both single-stranded and double-strandedportions, and may contain triple-stranded portions, but preferably isdouble-stranded DNA.

As used herein, a “therapeutically effective amount” refers to thatamount of the therapeutic agent sufficient to treat or manage a diseaseor disorder. A therapeutically effective amount may refer to the amountof therapeutic agent sufficient to delay or minimize the onset ofdisease, e.g., delay or minimize the spread of cancer. A therapeuticallyeffective amount may also refer to the amount of the therapeutic agentthat provides a therapeutic benefit in the treatment or management of adisease. Further, a therapeutically effective amount with respect to atherapeutic agent of the invention means the amount of therapeutic agentalone, or in combination with other therapies, that provides atherapeutic benefit in the treatment or management of a disease.

As used herein, the terms “prophylactic agent” and “prophylactic agents”refer to any agent(s) which can be used in the prevention of a disorder,or prevention of recurrence or spread of a disorder. A prophylacticallyeffective amount may refer to the amount of prophylactic agentsufficient to prevent the recurrence or spread of hyperproliferativedisease, particularly cancer, or the occurrence of such in a patient,including but not limited to those predisposed to hyperproliferativedisease, for example those genetically predisposed to cancer orpreviously exposed to carcinogens. A prophylactically effective amountmay also refer to the amount of the prophylactic agent that provides aprophylactic benefit in the prevention of disease. Further, aprophylactically effective amount with respect to a prophylactic agentof the invention means that amount of prophylactic agent alone, or incombination with other agents, that provides a prophylactic benefit inthe prevention of disease.

As used herein, the terms “prevent”, “preventing” and “prevention” referto the prevention of the recurrence or onset of one or more symptoms ofa disorder in a subject as result of the administration of aprophylactic or therapeutic agent.

As used herein, the term “in combination” refers to the use of more thanone prophylactic and/or therapeutic agents. The use of the term “incombination” does not restrict the order in which prophylactic and/ortherapeutic agents are administered to a subject with a disorder. Afirst prophylactic or therapeutic agent can be administered prior to(e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeksbefore), concomitantly with, or subsequent to (e.g., 5 minutes, 15minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks,4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) theadministration of a second prophylactic or therapeutic agent to asubject with a disorder.

“Effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toantibody dependent cell mediated cytotoxicity (ADCC), antibody dependentcell mediated phagocytosis (ADCP), and complement dependent cytotoxicity(CDC). Effector functions include both those that operate after thebinding of an antigen and those that operate independent of antigenbinding.

“Effector cell” as used herein is meant a cell of the immune system thatexpresses one or more Fc receptors and mediates one or more effectorfunctions. Effector cells include but are not limited to monocytes,macrophages, neutrophils, dendritic cells, eosinophils, mast cells,platelets, B cells, large granular lymphocytes, Langerhans' cells,natural killer (NK) cells, and may be from any organism including butnot limited to humans, mice, rats, rabbits, and monkeys.

“Fc ligand” as used herein is meant a molecule, preferably apolypeptide, from any organism that binds to the Fc region of anantibody to form an Fc-ligand complex. Fc ligands include but are notlimited to FcγRs, FcγRs, FcγRs, FcRn, C1q, C3, staphylococcal protein A,streptococcal protein G, and viral FcγR. Fc ligands may includeundiscovered molecules that bind Fc.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Amino Acid Sequence of Human IgG1 Hinge-Fc Region

FIG. 1 shows the amino acid sequence of the human IgG1 hinge-Fc region(SEQ ID NO:11). The amino acid residues shown in the figure, 1-232,correspond to amino acid residues 231 to 447 of the IgG heavy chainaccording to the numbering system of Kabat.

FIG. 2 SDS-Page Analysis of Recombinant Soluble FcγR

The purity of recombinant soluble FcγR proteins was assessed by 10%polyacrylamide gel electrophoresis. The gels were stained with Coomassieblue. Lane 1: purified recombinant soluble FcγRIIIA; Lane 2: molecularweight marker; Lane 3: molecular weight marker; Lane 4: purifiedrecombinant soluble FcγRIIB. The dashes refer to the molecular weight ofthe markers, from top to bottom, they correspond to a molecular weightof 98, 50, 36, and 22 KDa respectively.

FIG. 3 ELISA Assay of Recombinant Soluble FcγR

The direct binding of purified recombinant soluble FcγRIIIA toaggregated and monomeric IgG was determined using an ELISA assay.Binding of (▴) aggregated IgG with 3G8; (♦) Biotinylated IgG; (▪)aggregated IgG; (X) aggregated IgG with mouse IgG1.

FIGS. 4 A and B Characterization of FcγRIIIA Tetrameric Complex Using anELISA Assay

A. Soluble tetrameric FcγRIIIA complex binds soluble monomeric human IgGspecifically. Binding of soluble tetrameric FcγRIIIA to human IgG isblocked by 3G8 (♦), a mouse anti-FcγIIIA monoclonal antibody; the 4-4-20monoclonal antibody harboring the D265A mutation was not able to blockthe binding of soluble tetrameric FcγRIIIA to aggregated human IgG (Δ).

B. Binding of soluble tetrameric FcγRIIIA complex to soluble monomerichuman IgG (▪) is compared to the binding of monomeric soluble FcγRIIIAto soluble monomeric human IgG (♦).

FIGS. 5A-5E Characterization of FcγRIIIA Tetrameric Complex Using aMagnetic Bead Assay

A. FIG. 5A: FcγRIIIA Complex: two FcγRIIIA(filled shape) are joined by amonoclonal antibody DJ130c (1^(st) Ab); the anti-mouse F(ab)₂ isconjugated to PE (circle).

B. FACS analysis of FcγRIIIA bound to Fc coated beads: FIG. 5B: beadsalone; FIG. 5C: complex without FcγRIIIA; FIG. 5D: complex withFcγRIIIA; FIG. 5E: complex with FcγRIIIA and LNK16.

FIG. 6 Schematic Presentation of Fc Containing Constructs

A schematic diagram of the IgG1 Fc domains cloned into pYD1 ispresented. The open box represents the hinge-CH2-CH3 domains; parallelvertical lines represent the CH1 domain. In the case of the GIF206 and227 constructs; the N-terminal amino acids (SEQ ID NO:12 and SEQ IDNO:13, respectively) are shown. The underlined residues correspond tothe hinge region; the * represents the Xpress epitope tag; hatched boxesrepresent the Gly4-Ser linker, and the stippled boxes represent theAga2p gene.

FIGS. 7A-H FACS Analysis of the Fc Fusion Proteins on the Yeast CellWall

Cells were incubated with either a PE-conjugated polyclonal goatanti-human Fc antibody (FIGS. 6A-D) or with HP6017 (Sigma), a mouseanti-human IgG1 Fc (CH3) specific monoclonal antibody (FIGS. 6E-H). Aand E represent vector alone; Panels B and F represent the CH1—CH3construct; Panels C and G represent the GIF227; and Panels D and Hrepresent the GIF 206 construct.

FIGS. 8A-C Binding of Soluble Tetrameric FcγRIIIA to the SurfaceDisplayed Fc Fusion Proteins

Cells containing pYD1-CH1 (A); pYD-CH1-D265A (B); and pYD vector (C)were grown under conditions to express Aga2p fusion proteins on the cellsurface. Cells were incubated with FcγRIIIA at 0.15 mM, 7.5 mM, and 7.5mM, respectively, and analyzed by FACS.

FIG. 9 Characterization of the Binding of Soluble Tetrameric FcγRIIIA tothe Surface Displayed Fc Fusion Proteins

Binding of FcγRIIIA tetrameric complex to Fc fusion proteins on theyeast cell surface was analyzed. PE-conjugated FcγRIIIA tetramericcomplexes were pre-incubated with different concentrations of 3G8 (♦),LNK (▴) or an irrelevant isotype control (▪), and subsequently incubatedwith the yeast cells. Cells were analyzed by FACS for PE fluorescence.The percent cells that bound the FcγRIIIA tetrameric complex wereplotted on the y-axis.

FIG. 10 Example of Sort Gate for Selecting Fc Mutants With IncreasedBinding to FcγRIIIA

Cells were stained with PE-conjugated FcγRIIIA tetrameric complexes(y-axis) and anti-Fc-FITC conjugated antibody α-axis). Boxed arearepresents sort gate set to select ˜1.0% of the cell population.

FIGS. 11A-N FACS Analysis of Some of the Fc Mutants Identified Having anIncreased Affinity for FcγRIIIA Tetrameric Complexes

Individual clones harboring the pYD-CH1 plasmid containing independentFc mutations were amplified in selective media containing glucose,induced for Fc expression in selective media containing galactose, andsubsequently analyzed by FACs. FIGS. 10A and B represent cells harboringwild-type Fc; FIGS. 10C and D represent mutant #5; FIGS. 10E and Frepresent mutant #20; FIGS. 10G and H represent mutant #21; FIGS. 10 Iand J represent mutant #24; FIGS. 10K and L represent mutant #25; FIGS.10M and N represent mutant #27. Cells were stained with FcγRIIIAtetrameric complex (FIGS. 10 A, C, E, G, I, K, and M) or FcγRIIBtetrameric complex (FIGS. 10B, D, F, H, J, L, and N).

FIGS. 12 A-B Characterization of Fc Mutants in the 4-4-20 MonoclonalAntibody by ELISA

Fc domains from the pYD-CH1 plasmids were cloned into the heavy chain ofthe chimeric 4-4-20 monoclonal antibody. The 4-4-20 monoclonal antibodywas expressed in 293 cells and supernatants were collected. ELISA plateswere coated with fluoresceine conjugated BSA to capture the chimeric4-4-20 mutant antibodies. FcγRIIIA (A) and FcγRIIB (B) receptors werethen coated onto the ELISA plates to which the 4-4-20 monoclonalantibodies had been absorbed in order to determine the relativeaffinities of the variant receptors to the Fc domains. Mutants #15 and#29 were non-binding isolates included as controls.

FIG. 13 ADCC Activity of Mutants in the 4-4-20 Monoclonal Antibody

4-4-20 antibodies containing mutant Fc regions were assessed for theirADCC activity, and compared to the ADCC activity of a wild type 4-4-20antibody. The mutants analyzed are as follows: MGFc-10 (K288N, A330S,P396L), MGFc-26 (D265A), MGFc-27 (G316D, A378V, D399E), MGFc28 (N315I,A379M, D399E), MGFc29 (F243I, V379L, G420V), MGFc30 (F275V), MGFc-31(P247L, N421K), MGFc-32 (D280E, S354F, A431D, L441I), MGFc-33 (K317N,F423 deleted), MGFc-34 (F241L, E258G), MGFc-35 (R255Q, K326E), MGFc-36(K218R, G281D, G385R)

FIGS. 14 A and B ADCC Activity of Mutants in the HER2/neu HumanizedMonoclonal Antibody

A. Humanized HER2/neu monoclonal antibodies containing mutant Fc regionswere assessed for their ADCC activity and compared to the ADCC activityof a wild type Her2/neu antibody. The mutants analyzed are as follows:MGFc-5 (V379M), MGFc-9 (F243I, V379L), MGFc-10 (K288N, A330S, P396L),MGFc-13 (K334E, T359N, T366S), MGFc-27 (G316D, A378V, D399E).

B. ADCC activity of additional mutants in the context of the humanizedHer2/neu monoclonal antibody MGFc-37 (K248M), MGFc-39 (E293V Q295E,A327T), MGFc-38 (K392T, P396L), MGFc-41 (H268N, P396L), MGFc-23 (K334E,R292L), MGFc-44, MGFc-45. Two independent clones were tested for eachmutant.

FIG. 15 Capture of ch 4-4-20 Antibody on BSA-FITC Surface

6 μL of antibody at a concentration of approximately 20 μg/mL wasinjected at 5 μL/min over a BSA-fluoroscein isothiocyanate (FITC)surface. BIAcore sensogram of the binding of ch 4-4-20 antibodies withmutant Fc regions on the surface of the BSA-FITC immobilized sensor shipis shown. The marker was set on wild-type captured antibody response.

FIG. 16 Sensogram of Real Time Binding of FcγRIIIA to ch 4-4-20Antibodies Carrying Variant Fc Regions

Binding of FcγRIIIA to ch-4-4-20 antibodies carrying variant Fc regionswas analyzed at 200 nM concentration. Responses were normalized at thelevel of ch-4-4-20 antibody obtained for wild-type.

Mutants used were as follows: Mut 6 (S219V), Mut 10 (P396L, A330S,K288N); Mut 18 (K326E); Mut 14 (K334E, K288N); Mut 11 (R255L, F243L);Mut 16 (F372Y); Mut 19 (K334N, K246I).

FIGS. 17 A-H Analysis of Kinetic Parameters of FcγRIIIA Binding toAntibodies Carrying Variant Fc Regions

Kinetic parameters for FcγRIIIA binding to antibodies carrying variantFc regions were obtained by generating separate best fit curves for 200nM and 800 nM. Solid line indicates an association fit which wasobtained based on the k_(off) values calculated for the dissociationcurves in the 32-34 sec interval. K_(d) and k_(off) values represent theaverage from two concentrations.

FIG. 18 Sensogram of Real Time Binding of FcγRIIB-Fc Fusion Proteins toAntibodies Carrying Variant Fc Regions

Binding of FcγRIIB-Fc fusion proteins to ch-4-4-20 antibodies carryingvariant Fc regions was analyzed at 200 nM concentration. Responses werenormalized at the level of ch-4-4-20 antibody obtained for wild type.

FIGS. 19 A-C Analysis of Kinetic Parameters FcγRIIB-Fc Fusion Proteinsto Antibodies Carrying Variant Fc Regions

Kinetic parameters for FcγRIIB-Fc binding to antibodies carrying variantFc regions were obtained by generating separate best fit curves for 200nM and 800 nM. Solid line indicates an association fit which wasobtained based on the k_(off) values calculated for the dissociationcurves in the 32-34 sec. interval. K_(d) and K_(off) values representthe average from two concentrations.

Mutants used were as follows: Mut 6 (S219V), Mut 10 (P396L, A330S,K288N); Mut 18 (K326E); Mut 14 (K334E, K288N); Mut 11 (R255L, F243L);Mut 16 (F372Y); Mut 19 (K334N, K246I).

FIG. 20 Ratios of K_(off) (Wt)/K_(off) (Mut) for FcγRIIIA-Fc PlottedAgainst ADCC Data

Numbers higher than one show a decreased dissociation rate for FcγRIIIAbinding and increased dissociation rate for FcγRIIB-Fc binding relativeto wild-type. Mutants in the box have lower off rate for FcγRIIIAbinding and higher off rate for FcγRIIB-Fc binding.

FIG. 21 Competition with Unlabeled FcγRIIIA

A kinetic screen was implemented to identify Fc region mutants withimproved K_(off) rates for binding FcγRIIIA. A library of Fc regionvariants containing P396L mutation was incubated with 0.1 μMbiotinylated FcγRIIIA-Linker-AVITAG™ peptide for one hour and thenwashed. Subsequently 0.8 μM unlabeled FcγRIIIA was incubatd with thelabeled yeast for different time points. Yeast was spun down andunlabeled FcγRIIIA was removed, Receptor bound yeast was stained with SA(streptavidin):PE (phycoerythrin) for FACS analysis.

FIGS. 22 A-C FACS Analysis Based on the Kinetic Screen

Based on the calculated K_(off) from the data presented in FIG. 20, aone minute time point selection was chosen. A 10-fold excess of librarywas incubated with 0.1 μM biotinylated FcγRIIIA-Linker-AVITAG™ peptidemonomer; cells were washed and incubated with unlabeled ligand for oneminute; then washed and labeled with SA:PE. The cells were then sortedby FACS, selecting the top 0.3% binders. The non-selected P396L librarywas compared to the yeast cells selected for improved binding by FACS.The histograms show the percentage of cells that are costained with bothFcγRIIIA/PE and goat anti-human Fc/FITC.

FIGS. 23 A-F and G-L Selection Based on Solid Phase Depletion of FcγRIIBFc Binders

FIGS. 23 A-F. The P396L library was screened based on FcγRIIB depletionand FcγRIIIA selection using magnetic beads. The FcγRIIB depletion bymagnetic beads was repeated 5 times. The resulting yeast population wasanalyzed and found to show greater than 50% cell staining with goatanti-human Fc and a very small percentage of cells stained withFcγRIIIA. Subsequently cells were selected twice by FACS using 0.1 μMbiotinylated FcγRIIIA linker-AVITAG™ peptide. Yeast cells were analyzedfor both FcγRIIIA and FcγRIIB binding after each sort and compared towild type binding.

FIGS. 23 G-L. Fc Mutants were selected from the FcγRIIB depleted yeastpopulation using biotinylated FcγRIIIA 158F linker avitag monomer as aligand. The sort gate was set to select the top 0.25% FcγRIIIA 158Fbinders. The resulting enriched population was analyzed by FACS forbinding to the different FcγRIIIA (158F and 158V), FcγRIIIB and FcγRIIA(131R).

FIG. 24 Relative Rates of SKBR3Target Cell Lysis Mediated by Chimeric4D5 Harboring Fc Mutants

Relative rates of lysis was calculated for each Fc mutant tested. Lysisrates for 4D5 antibody with Fc mutants were divided by the rate of lysismediated by wild type 4D5 antibody. Data from at least 2 independentassays were averaged and plotted on the histogram. For each Fc mutantdata from two different antibody concentrations are shown. The antibodyconcentrations were chosen to flank the point along the curve at whichlysis was ˜50%.

FIG. 25 Relative Rates of Daudi Cell Lysis Mediated by Chimeric 2H7Harboring Fc Mutants

Relative rates of lysis was calculated for each Fc mutant tested. Lysisrates for 2H7 antibody with Fc mutants were divided by the rate of lysismediated by wild type 2H7 antibody. Data from at least 1-2 independentassays were averaged and plotted on the histogram. For each Fc mutant,data from two different antibody concentrations are shown The antibodyconcentrations were chosen based on the point along the curve at whichlysis was ˜50%.

FIG. 26 Scheme for Library Production.

DNA strands are represented. Forward arrows represent primers containingmutant codons. Reverse arrow represent reverse gene specific oligo.

FIG. 27 Strategy for Production of Libraries by Build a Gene Protocol.

The rectangular boxes represent the hinge, CH2, and CH3 domains,respectively. The short black lines represent the double stranded oligoswith 5′ overhangs.

FIG. 28 Novel Fc Mutants Improve PBMC Mediated ADCC in SKBR3Cells.

The plot shows linear regression analysis of a standard ADCC assay.Antibody was titrated over 3 logs using an effector to target ratio of75:1. % lysis=(Experimental release—SR)/(MR−SR)*100.

FIG. 29 Novel Fc Mutants Improve PBMC Mediated ADCC in Daudi Cells.

The plot shows linear regression analysis of a standard ADCC assay.Antibody was titrated over 3 logs using an effector to target ratio of75:1. % lysis=(Experimental release—SR)/(MR−SR)*100.

FIGS. 30A-30O Fc Receptor Profiles Via FACS Upon Cytokine Treatment ofMonocytes.

Cytokine treatment of monocytes increases low affinity Fc receptorexpression Elutriated monocytes were cultured using specific cytokinesin serum free media. Fc receptor profiles were assayed using FACS.

FIG. 31 Improved Tumor Cell Killing Using Fc Mutants inMacrophage-Derived Monocytes Based ADCC.

Ch4D5 MAb concentration over 2 logs was tested using effector:targetratio of 35:1. Percent lysis was calculated as in FIG. 28.

FIG. 32 Complement Dependent Cytotoxicity Assay Flow Chart.

The flow chart summarizes the CDC assays used.

FIG. 33 Complement Dependent Cytotoxicity Activity

Fc mutants that show enhanced binding to FcγRIIIA also showed improvedcomplement activity. Anti-CD20 ChMAb over 3 orders of magnitude wastitrated. Percent lysis was calculated as in as in FIG. 28.

FIG. 34 Decision Tree for Selection of Fc Mutants

An exemplary protocol for selecting Fc mutants. In the Figure, the term“Rituxan” denotes RITUXAN™ anti-CD20 antibody.

FIGS. 35A-35C C1q Binding to 2B6 Antibody

FIG. 35A. The diagram depicts the BIAcore format for analysis of 2B6binding to the first component of the complement cascade.

FIGS. 35B-35C. Sensogram of real time binding of 2B6 antibody carryingvariant Fc regions to C1q.

FIGS. 36 A-D C1q Binding to 2B6 Mutant Antibody.

Sensogram of real time binding of 2B6 mutants to C1q (3.25 nM). Mutantsdepicted at MgFc51 (Q419H, P396L); MgFc51/60 in Panel A; MgFc55 andMgFc55/60 (Panel B), MgFc59 and MgFc59/60 (Panel C); and MgFc31/60(Panel D).

FIGS. 37 A-D Fc Variants with Decreased Binding to FcγRIIB

Binding of FcR to ch4D5 antibodies to compare effect of D270E (60) onR255L, P396L double mutant (MgFc55). K_(D) was analyzed at differentconcentrations of FcR; 400 nM CD16A 158V; 800 nM CD16A 158F; 200 nMCD32B; 200 nM CD32A 131H. Analysis was performed using separate K_(D)using Biacore 3000 software.

FIGS. 38 A-D Kinetic Characteristics of 4D5 Mutants Selected fromFcγRIIB Depletions/FcγRIIAH131 Selection

Binding of FcR to ch4D5 antibodies carrying different Fc mutationsselected by CD32B depletion and CD32A H131 screening strategy. K_(D) wasanalyzed at different concentrations of FcR; 400 nM CD16A 158V; 800 nMCD16A 158F; 200 nM CD32B; 200 nM CD32A 131H. Analysis was performedusing separate K_(D) using Biacore 3000 software.

FIG. 39 Plot of MDM ADCC Data Against the K_(OFF) Determined for CD32A131H Binding as Determined by Biacore.

The mutants are as follows: MgFc 25 (E333A, K334A, S298A); MgFc68(D270E); MgFc38 (K392T, P396L); MgFc55 (R255L, P396L); MgFc31 (P247L,N421K); MgFc59(K370E, P396L).

FIG. 40 A-D FcγR Binding to 4D5 Mutant Antibody, Triple Mutation

Sensogram of real time binding of 4D5 mutants to FcγRIII3A (CD16Z V¹⁵⁸,panel A, and CD16A F¹⁵⁸, panel B), FcγRIIB (CD32B, panel C) and FcγRIIA(CD32A H¹³¹, panel D). Mutants depicted are MgFc31/60 (P247L; N421K;D270E) (short dashed line), MgFc71 (D270E; G316D; R416G) (solid line)and AAA (E333A; K334A; S298A) (long dashed line). The binding ofwild-type 4D5 (dash-dot line) is also provided.

FIG. 41A-D FcγR Binding to 4D5 Mutant Antibody, Quadruple Mutation

Sensogram of real time binding of 4D5 mutants to FcγRIII3A (CD16Z V¹⁵⁸,panel A, and CD16A F¹⁵⁸, panel B), FcγRIIB (CD32B, panel C) and FcγRIIA(CD32A H¹³¹, panel D). Mutants depicted are MgFc55/60/F243L (R255L;P396L; D270E; F243L), MgFc38/60/F243L (K392T; P396L; D270E; F243L) andAAA (E333A; K334A; S298A). The binding of wild-type 4D5 is alsoprovided.

FIG. 42 A-E Binding of 4D5 Variant 31/60 to HT29 Cells

FACS analysis was used to characterize the binding of monoclonalanti-HER2/neu antibody ch4D5, variant 31/60 (P247L; N421K; D270E), toHT29 cells (low expression of HER2/neu). Incubation with primaryantibody was at 10 μg/ml (A), 1 μg/ml (B), 0.1 μg/ml (C), 0.001 μg/ml(D), or 0.001 μg/ml (E). Wild-type ch4D5 and SYNAGIS® (palivizumab) wereused as controls. PE-conjugated polyclonal F(ab)₂ goat anti-humanFCγRwas used as the secondary antibody.

FIG. 43 A-B ADCC Activity of Mutants in the Anti-HER2/neu Antibody,ch4D5

CH4D5 antibodies containing mutant Fc regions were assessed for theirADCC activity and compared to the ADCC activity of wild type ch4D5.SKBR3 (high expression of HER2/neu) and HT29 (low expression ofHER2/neu) cells lines were used as targets (panels A and B,respectively). Effector to target ratio (E:T ratio) was 50:1 with 18 hincubation. Mutants analyzed were MGFc59/60 (K370E; P396L; D270E),MGFc55/60 (R255L; P396L; D270E), MGFc51/60 (Q419H; P396L; D270E),MGFc55/60/F243L (R255L; P396L; D270E; F243L); MGFc74/P396L (F243L;R292P; V3051; P396L).

FIG. 44 A-B ADCC Activity of Mutants in the Anti-HER2/neu Antibody,ch4D5

Ch4D5 antibodies containing mutant Fc regions were assessed for theirADCC activity and compared to the ADCC activity of wild type ch4D5.SKBR3 (high expression of HER2/neu) and HT29 (low expression ofHER2/neu) cells lines were used as targets (panels A and B,respectively). Effector to target ratio (E:T ratio) was 75:1 with 18 hincubation. Mutants analyzed were MgFc31/60 (P247L; N421K; D270E) andMgFc71 (D270E; G316D; R416G).

FIGS. 45A-45H Binding of Mutants in the Monoclonal Anti-CD32B AntibodyCh2B6 to Daudi Cells and Ramos Cells

FACS analysis was used to characterize the binding of monoclonalanti-CD32B antibody ch2B6 variant 31/60 (P247L; N421K; D270E), variant71 (D270E; G316D; R416G) and variant 59/60 (K370E; P396L; D270E) toeither Daudi cells (high expression of CD32B) or Ramos cells (lowexpression of CD32B). Incubation with primary antibody was at 5 μg/ml,0.5 μg/ml, 50 ng/ml, or 5 ng/ml. Wild-type ch2B6 and IgG (SYNAGIS) wereused as controls. PE-conjugated polyclonal F(ab)₂ goat anti-humanFCγRwas used as the secondary antibody.

FIG. 46 A-B ADCC Activity of Mutants in the Anti-CD32B Antibody, ch2B6

Ch2B6 antibodies containing mutant Fc regions were assessed for theirADCC activity and compared to the ADCC activity of wild type 2B6. TheRamos cell line (low expression of CD32B) was used as target. Effectorto target ratio (E:T ratio) was 75:1 with 18 h incubation. Mutantsanalyzed were variant 31/60 (P247L; N421K; D270E) and ch2B6 N297Q(aglycoslyated Fc, no FcR binding) (panel A); and MGFc51/60/F243L(Q419H; P396L; D270E; F243L); MGFc55/60/F243L (R255L; P396L; D270E;F243L) and MGFc38/60/F243L (K392T; P396L; D270E; F243L) (panel B).Wild-type ch2B6 or RITUXAN™ (rituximab) were used as controls.

FIG. 47 A-C CDC Activity of Mutants in the Anti-CD32B Antibody, ch2B6

Ch2B6 antibodies containing mutant Fc regions were assessed for theirCDC activity and compared to the CDC activity of wild type ch2B6. BL41(a Burkitt's lymphoma cell line) (panel A and B) and Ramos (lowexpression of CD32B) (panel C) cells lines were used as targets.Effector to target ratio (E:T ratio) was 75:1 with 18 h incubation.Mutants analyzed were MgFc31/60 (P247L; N421K; D270E) and,MGFc55/60/Y300L (R255L; P396L; D270E; Y300L) (panel A); MgFc71 (D270E;G316D; R416G), MGFc51/60/F243L (Q419H; P396L; D270E; F243L), andMGFc55/60/F243L (R255L; P396L; D270E; F243L) (panel B); and MgFc31/60(P247L; N421K; D270E) (panel C). Wild-type ch2B6, wild-type humanizedch2B6 (hu2B6 wt) or RITUXAN™ (rituximab) were used as controls.

FIG. 48 A-B ADCC Activity of Mutants in the Anti-CD32B Antibody, ch2B6

Ch2B6 antibodies containing mutant Fc regions were assessed for theirADCC activity and compared to the ADCC activity of wild type ch2B6. TheDaudi cell line (high expression of CD32B) was used as target. Effectorto target ratio (E:T ratio) was 75:1 with 18 h incubation. Mutantsanalyzed were MgFc31/60 (P247L; N421K; D270E), ch2B6 Ag (N297Q;aglycoslyated Fc, no FcR binding) and MgFc71 (D270E; G316D; R416G)(panel A); and MGFc55/60/F243L (R255L; P396L; D270E; F243L),MGFc51/60/F243L (Q419H; P396L; D270E; F243L) and MGFc38/60/F243L (K392T;P396L; D270E; F243L) (panel B). Wild-type ch2B6 or RITUXAN™ (rituximab)were used as controls.

FIG. 49 A-B FACS Analysis of the Binding of the Anti-CD32B Antibody,ch2B6, and the Anti-CD20 Antibody, Rituxan™, to a Transgenic Cho CellLine.

Cho cells were engineered to express both recombinant CD32B andrecombinant CD20 on the cell surface. Following incubation andamplification in selective media, cells were analyzed by FACS. Cellswere incubated in either FITC-conjugated wild-type 2B6 (A) orFITC-conjugated Rituxan™ (B).

FIG. 50 A-B ADCC Activity of Mutants in the Anti-CD20 Antibody, Rituxan™

RITUXAN™ anti-CD20 antibodies containing mutant Fc regions were assessedfor their ADCC activity and compared to the ADCC activity of wild typeRITUXAN™ anti-CD20 and ch2B6. A Cho cell line engineered to express bothCD32B and CD20 was used as target. Effector to target ratio (E:T ratio)was 75:1 with 18 h incubation. Figure A shows the ADCC activity of wildtype ch2B6 and RITUXAN™ anti-CD20. Figure B shows a comparison of theADCC activity of wild type RITUXAN™ anti-CD20 and RITUXAN™ anti-CD20comprising mutation variant MGFc55/60 (R255L; P396L; D270E).

FIG. 51A-E Comparison of Binding Affinity and Kinetic Characteristics ofch2B6 Mutants

FACS analysis was used to characterize the binding of mutant ch2B6antibodies to Ramos cells (low expression of CD32B). Data were comparedto a BIAcore analysis of the k_(off) for the same variant antibodies.Mutants analyzed were MgFc55 (R255L; P396L), MgFc55/60 (R255L; P396L;D270E) and MgFc55/60/F243L (R255L; P396L; D270E; F243L). Wild-type ch2B6was used as control. Incubation with primary antibody was at 10 μg/ml(A), 1 μg/ml (B), 0.1 ng/ml (C), or 0.01 ng/ml (D). PE-conjugatedpolyclonal F(ab)₂ goat anti-humanFCγR was used as the secondaryantibody.

FIG. 52 A-C Binding of Activating Receptor CD16A to Ramos CellsOpsonized with Mutant ch2B6 Antibody

FACS analysis was used to characterize the binding of activatingreceptor CD16A to Ramos cells opsonized with mutant ch2B631/60 antibody(P247L; N421K; D270E). Opsonization with wild-type ch2B6, hu2B6YA(humanized 2B6 with YA substitution at positions 50,51 of antibodylight-chain—eliminates glycosylation at position 50 of the light-chainprotein), or antibody-free buffer was used as a control. PE-conjugatedpolyclonal F(ab)₂ goat anti-humanFCγR was used as the secondaryantibody.

FIG. 53 A-J Binding of Mutants in the Monoclonal Anti-CD32B Antibodych2B6 to Daudi Cells

FACS analysis was used to characterize the binding of monoclonalanti-CD32B antibody ch2B6 variant 31/60 (P247L; N421K; D270E), hu2B6YA(humanized 2B6 with YA substitution at positions 50,51 of antibodylight-chain—eliminates glycosylation at position 50 of the light-chainprotein) or hu2B6YA31/60 to Daudi cells (high expression of CD32B).Wild-type ch2B6, hu2B6, SYNAGIS® (palivizumab) and ch2B6 Agly (N297Q;aglycoslyated Fc, no FcR binding) were used as controls. Incubation withprimary antibody was at either 37° C. (panels A-E) or 4° C. (panels F-J)for 0.5 h and at a concentration of 10 μg/ml (A, F), 1 μg/ml (B, G), 0.1ng/ml (C, H), 0.01 μg/ml (D, I), or 0.001 μg/ml (E, J). PE-conjugatedpolyclonal F(ab)₂ goat anti-humanFCγR was used as the secondaryantibody.

FIG. 54 A-E Binding of Mutants in the Monoclonal Anti-CD32B Antibodych2B6 to EL-4/CD32B Cells

FACS analysis was used to characterize the binding of monoclonalanti-CD32B antibody ch2B6 variant 31/60 (P247L; N421K; D270E), hu2B6YA(humanized 2B6 with YA substitution at positions 50,51 of antibodylight-chain—eliminates glycosylation at position 50 of the light-chainprotein) or hu2B6YA31/60 to EL-4/CD32B cells. Wild-type ch2B6, hu2B6,SYNAGIS® (palivizumab) and ch2B6 Agly (N297Q; aglycoslyated Fc, no FcRbinding) were used as controls. Incubation with primary antibody was at37° C. for 0.5 h and at a concentration of 10 μg/ml (A), 1 μg/ml (B),0.1 μg/ml (C), 0.01 μg/ml (D), or 0.001 μg/ml (E). PE-conjugatedpolyclonal F(ab)₂ goat anti-humanFCγR was used as the secondaryantibody.

FIG. 55 A-J Binding of Mutants in the Monoclonal Anti-CD32B Antibodych2B6 to Ramos Cells

FACS analysis was used to characterize the binding of monoclonalanti-CD32B antibody ch2B6 variant 31/60 (P247L; N421K; D270E), hu2B6YA(humanized 2B6 with YA substitution at positions 50,51 of antibodylight-chain—eliminates glycosylation at position 50 of the light-chainprotein) or hu2B6YA31/60 to Ramos cells (low expression of CD32B).Wild-type ch2B6, hu2B6, SYNAGIS® (palivizumab) and ch2B6 Agly (N297Q;aglycoslyated Fc, no FcR binding) was used as control. Incubation withprimary antibody was at either 37° C. (panels A-E) or 4° C. (panels F-J)for 0.5 h and at a concentration of 10 μg/ml (A, F), 1 μg/ml (B, G), 0.1ng/ml (C, H), 0.01 μg/ml (D, I), or 0.001 μg/ml (E, J). PE-conjugatedpolyclonal F(ab)₂ goat anti-humanFCγR was used as control.

FIG. 56 ADCC Activity of Mutants in the Anti-CD32B Antibody, ch2B6

Ch2B6 antibodies containing mutant Fc regions were assessed for theirADCC activity and compared to the ADCC activity of wild type ch2B6. TheRamos cell line (low expression of CD32B) was used as target. Effectorto target ratio (E:T ratio) was 75:1 with 18 h incubation. Mutantsanalyzed were MGFc31/60 (P247L; N421K; D270E), MGFc51/60 (Q419H; P396L;D270E), MGFc55/60 (R255L; P396L; D270E); hu2B6YA (humanized 2B6 with YAsubstitution at positions 50,51 of antibody light-chain—eliminatesglycosylation at position 50 of the light-chain protein), hu2B6YAMGFc51/60; hu2B6YA MGFc31/60 and hu2B6YA MGFc55/60. Wild-type ch2B6,hu2B6, RITUXAN™ (rituximab) and ch2B6 Agly (N297Q; aglycoslyated Fc, noFcR binding) were used as controls.

FIG. 57 CDC Activity of Mutants in the Anti-CD32B Antibody, ch2B6

Ch2B6 antibodies containing mutant Fc regions were assessed for theirCDC activity and compared to the CDC activity of wild type ch2B6. TheRamos cell line (low expression of CD32B) was used as target. Effectorto target ratio (E:T ratio) was 75:1 with 18 h incubation. Mutantsanalyzed were MGFc31/60 (P247L; N421K; D270E), MGFc51/60 (Q419H; P396L;D270E), MGFc55/60 (R255L; P396L; D270E); hu2B6YA (humanized 2B6 with YAsubstitution at positions 50,51 of antibody light-chain—eliminatesglycosylation at position 50 of the light-chain protein), hu2B6YAMGFc51/60; hu2B6YA MGFc31/60 and hu2B6YA MGFc55/60. Wild-type ch2B6 andRITUXAN™ anti-CD20 antibodies were used as controls.

FIG. 58 A-F ADCC Activity of Modified Rituximab Antibodies in HumanPatients Treated with Rituximab

Rituximab antibodies containing mutant Fc regions were assessed fortheir ADCC activity and compared to the ADCC activity of wild typerituximab. Patient derived cells were used as target. Effector to targetratio (E:T ratio) was 30:1 and 10:1. Mutants analyzed wereMGFc55/60/300L (R255L; P396L; D270E; Y300L); MGFc51/60 (Q419H; P396L;D270E); MGFc52/60 (V240A; P396L; D270E); MGFc59/60 (K370E; P396L;D270E); MGFc38/60 (K392T; P396L; D270E); MGFc59 (K370E; P396L); MGFc51(Q419H; P396L); MGFc31/60 (P247L; N421K; D270E); MGFc55/292G (R255L;P396L; D270E; R292G).

5. DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to engineering molecules, preferablypolypeptides, and more preferably immunoglobulins (e.g., antibodies) toconfer one or more effector function activities to the molecule, whicheffector functions the parent molecule does not have or has at lowlevels (e.g., not detectable in in vitro and/or in vivo assays known inthe art). In particular, the modified molecules, e.g., antibodies, ofthe invention comprise a variant Fc region, having one or more aminoacid modifications (e.g., substitutions, but also including insertionsor deletions) in one or more regions, which modifications confer atleast one effector function. In particular, the modifications alter theaffinity and avidity of the variant Fc region for an FcγR (e.g.,activating FcγRs or inhibitory FcγRs) and thereby altering the activityof one or more effector functions. In other embodiments, themodifications confer homo-oligomerization activity to the parent Fcregion such that oligomerization of the modified antibody cross-linkscell-surface antigens, resulting in apoptosis, negative-growthregulation or cell killing. Effector function activities that may beconferred include, but are not limited to, antibody-dependent cellmediated cytotoxicity (ADCC), antibody-dependent phagocytosis,phagocytosis, opsonization, opsonophagocytosis, cell binding, rosetting,and complement dependent cell mediated cytotoxicity (CDC). In someembodiments of the invention, the modifications alter the affinity ofthe variant Fc region such that the variant Fc regions oligomerize andhomo-oligomers of the modified antibody are formed. In certainembodiments of the invention, the engineered molecule is not ananti-CD20 antibody, more particularly, does not compete for CD20 bindingwith rituximab or is not rituximab.

The present invention also relates to molecules (e.g., antibodies)comprising a variant Fc region having one or more amino acidmodifications (e.g., substitutions, deletions, insertions) in one ormore portions, which modifications increase the affinity and avidity ofthe variant Fc region for an FcγR (including activating and inhibitoryFcγRs). In some embodiments, said one or more amino acid modificationsincrease the affinity of the variant Fc region for FcγRIIIA and/orFcγRIIA. In another embodiment, the variant Fc region furtherspecifically binds FcγRIIB with a lower affinity than does the Fc regionof the comparable parent antibody (i.e., an antibody having the sameamino acid sequence as the antibody of the invention except for the oneor more amino acid modifications in the Fc region). In some embodiments,such modifications increase the affinity of the variant Fc region forFcγRIIIA and/or FcγRIIA and also enhance the affinity of the variant Fcregion for FcγRIIB relative to the parent antibody. In otherembodiments, said one or more amino acid modifications increase theaffinity of the variant Fc region for FcγRIIIA and/or FcγRIIA but do notalter the affinity of the variant Fc regions for FcγRIIB relative to theFc region of the parent antibody. In another embodiment, said one ormore amino acid modifications enhance the affinity of the variant Fcregion for FcγRIIIA and FcγRIIA but reduce the affinity for FcγRIIBrelative to the parent antibody. Increased affinity and/or avidityresults in detectable binding to the FcγR or FcγR-related activity incells that express low levels of the FcγR when binding activity of theparent molecule (without the modified Fc region) can not be detected inthe cells. In other embodiments, the modified molecule exhibitsdetectable binding in cells which express non-FcγR receptor targetantigens at a density of 30,000 to 20,000 molecules/cell, at a densityof 20,000 to 10,000 molecules/cell, at a density of 10,000 to 5,000molecules/cell, at a density of 5,000 to 1,000 molecules/cell, at adensity of 1,000 to 200 molecules/cell or at a density of 200molecules/cell or less (but at least 10, 50, 100 or 150 molecules/cell).

In another embodiment, said one or more modifications to the amino acidsof the Fc region reduce the affinity and avidity of the antibody for oneor more FcγR receptors. In a specific embodiment, the inventionencompasses antibodies comprising a variant Fc region, wherein saidvariant Fc region comprises at least one amino acid modificationrelative to a wild type Fc region, which variant Fc region only bindsone FcγR, wherein said FcγR is FcγRIIIA. In another specific embodiment,the invention encompasses antibodies comprising a variant Fc region,wherein said variant Fc region comprises at least one amino acidmodification relative to a wild type Fc region, which variant Fc regiononly binds one FcγR, wherein said FcγR is FcγRIIA.

Preferably, the binding properties of the molecules of the invention arecharacterized by in vitro functional assays for determining one or moreFcγR mediator effector cell functions (See Section 5.2.7). Theaffinities and binding properties of the molecules, e.g., antibodies, ofthe invention for an FcγR can be determined using in vitro assays(biochemical or immunological based assays) known in the art fordetermining antibody-antigen or Fc-FcγR interactions, i.e., specificbinding of an antigen to an antibody or specific binding of an Fc regionto an FcγR, respectively, including but not limited to ELISA assay,surface plasmon resonance assay, immunoprecipitation assays (See Section5.2.1). In most preferred embodiments, the molecules of the inventionhave similar binding properties in in vivo models (such as thosedescribed and disclosed herein) as those in in vitro based assays.However, the present invention does not exclude molecules of theinvention that do not exhibit the desired phenotype in in vitro basedassays but do exhibit the desired phenotype in vivo.

In some embodiments, the molecules of the invention comprising a variantFc region comprise at least one amino acid modification in the CH3domain of the Fc region, which is defined as extending from amino acids342-447. In other embodiments, the molecules of the invention comprisinga variant Fc region comprise at least one amino acid modification in theCH2 domain of the Fc region, which is defined as extending from aminoacids 231-341. In some embodiments, the molecules of the inventioncomprise at least two amino acid modifications, wherein one modificationis in the CH3 region and one modification is in the CH2 region. Theinvention further encompasses amino acid modification in the hingeregion. In a particular embodiment, the invention encompasses amino acidmodification in the CH1 domain of the Fc region, which is defined asextending from amino acids 216-230.

In particularly preferred embodiments, the invention encompassesmolecules comprising a variant Fc region wherein said variant confers orhas an increased ADCC activity and/or an increased binding to FcγRIIA(CD32A), as measured using methods known to one skilled in the art andexemplified herein. The ADCC assays used in accordance with the methodsof the invention may be NK dependent or macrophage dependent.

The Fc variants of the present invention may be combined with other Fcmodifications known in the art. The invention encompasses combining anFc variant of the invention with other Fc modifications to provideadditive, synergistic, or novel properties to the modified antibody.Preferably, the Fc variants of the invention enhance the phenotype ofthe modification with which they are combined. For example, if an Fcvariant of the invention is combined with a mutant known to bindFcγRIIIA with a higher affinity than a comparable wild type Fc region;the combination with a mutant of the invention results in a greater foldenhancement in FcγRIIIA affinity.

The Fc variants of the present invention may be combined with anymodifications in the art such as those disclosed in Table 2 below.

TABLE 2 Substitution(s) V264A V264I/N297D/I332E V264L Y296D/N297D/I332EV264I Y296E/N297D/I332E F241W Y296N/N297D/I332E F241L Y296Q/N297D/I332EF243W Y296H/N297D/I332E F243L Y296T/N297D/I332E F241L/F243L/V262I/V264IN297D/T299V/I332E F241W/F243W N297D/T299I/I332E F241W/F243W/V262A/V264AN297D/T299L/I332E F241L/V262I N297D/T299F/I332E F243L/V2641N297D/T299H/I332E F243L/V262I/V264W N297D/T299E/I332EF241Y/F243Y/V262T/V264T N297D/A330Y/I332E F241E/F243R/V262E/V264RN297D/S298A/A330Y/I332E F241E/F243Q/V262T/V264E S239D/A330Y/I332EF241R/F243Q/V262T/V264R S239N/A330Y/I332E F241E/F243Y/V262T/V264RS239D/A330L/I332E L328M S239N/A330L/I332E L328E V264I/S298A/I332E L328FS239D/S298A/I332E I332E S239N/S298A/I332E L328M/I332E S239D/V264I/I332EP244H S239D/V264I/S298A/I332E P245A S239D/V264I/A330L/I332E P247V T256AW313F K290A P244H/P245A/P247V D312A P247G *K326A V264I/I332E S298AF241E/F243R/V262E/V264R/I332E E333A F241E/F243Q/V262T/V264E/I332E K334AF241R/F243Q/V262T/V264R/I332E E430A F241E/F243Y/V262T/V264R/I332E T359AS298A K360A S298A/I332E E430A S298A/E333A/K334A K320M S239E/I332E K326SS239Q/I332E K326N S239E K326D D265G K326E D265N K334Q S239E/D265G K334ES239E/D265N K334M S239E/D265Q K334H Y296E K334V Y296Q K334L S298T A330KS298N T335K T299I A339T A327S E333A, K334A A327N T256A, S298AS267Q/A327S T256A, D280A, S298A, T307A S267L/A327S S298A, E333A, K334AS298A, K334A A327L S298A, E333A P329F T256A A330L K290A A330Y K326AI332D R255A N297S E258A N297D S267A N297S/I332E E272A N297D/I332E N276AN297E/I332E D280A D265Y/N297D/I332E E283A D265Y/N297D/T299L/I332E H285AD265F/N297E/I332E N286A L328I/I332E P331A L328Q/I332E S337A I332N H268AI332Q E272A V264T E430A V264F A330K V240I R301M V263I H268N V266I H268ST299A E272Q T299S N286Q T299V N286S N325Q N286D N325L K290S N325I K320MS239D K320Q S239N K320E S239F K320R S239D/I332D K322E S239D/I332E K326SS239D/I332N K326D S239D/I332Q K326E S239E/I332D A330K S239E/I332N T335ES239E/I332Q S267A, E258A S239N/I332D S267A, R255A S239N/I332E S267A,D280A S239N/I332N S267A, E272A S239N/I332Q S267A, E293A S239Q/I332DS267A, E258A, D280A, R255A S239Q/I332N P238A S239Q/I332Q D265A K326EE269A Y296D D270A Y296N N297A F241Y/F243Y/V262T/V264T/ P329A N297D/I332EA330Y/I332E A327Q V264I/A330Y/I332E S239A A330L/I332E E294AV264I/A330L/I332E Q295A L234D V303A L234E K246A L234N I253A L234Q T260AL234T K274A L234H V282A L234Y K288A L234I Q311A L234V K317A L234F E318AL235D K338A L235S K340A L235N Q342A L235Q R344A L235T E345A L235H Q347AL235Y R355A L235I E356A L235V M358A L235F K360A S239T N361A S239H Q362AS239Y Y373A V240A S375A V240T D376A V240M E380A V263A E382A V263T S383AV263M N384A V264M Q386A V264Y E388A V266A N389A V266T N390A V266M Y391AE269H K392A E269Y L398A E269F S400A E269R D401A Y296S D413A Y296T K414AY296L S415A Y296I R416A A298H Q418A T299H Q419A A330V N421A A330I V422AA330F S424A A330R E430A A330H H433A N325D N434A N325E H435A N325A Y436AN325T T437A N325V Q438A N325H K439A L328D/I332E S440A L328E/I332E S442AL328N/I332E S444A L328Q/I332E K447A L328V/I332E K246M L328T/I332E K248ML328H/I332E Y300F L328I/I332E A330Q L328A K338M I332T K340M I332H A378QI332Y Y391F I332A S239E/V264I/I332E S239Q/V264I/I332ES239E/V264I/A330Y/I332E S239E/V264I/S298A/A330Y/I332E S239D/N297D/I332ES239E/N297D/I332E S239D/D265V/N297D/I332E S239D/D265I/N297D/I332ES239D/D265L/N297D/I332E S239D/D265F/N297D/I332E S239D/D265Y/N297D/I332ES239D/D265H/N297D/I332E S239D/D265T/N297D/I332E

In other embodiments, the Fc variants of the present invention may becombined with any of the known Fc modifications in the art such as thosedisclosed in Tables 3 A and B below.

TABLE 3A Starting Position Position Position Position Position Variant300 298 296 295 294 Y3001 + → — S298N, S298V, Y296P, Y296F, Q295K,Q295L, E294N, E294A, S298D, S298P, or N276Q. or Q295A. E294Q, or S298A,S298G, E294D. S298T, or S298L. Y300L + → — S298N, S298V, Y296P, Y296F,Q295K, Q295L, E294N, E294A, S298D, S298P, or N276Q. or Q295A. E294Q, orS298A, S298G, E294D. S298T, or S298L. S298N + → Y3001, Y300L, — Y296P,Y296F, Q295K, Q295L, E294N, E294A, or Y300F. or N276Q. or Q295A. E294Q,or E294D. S298V + → Y3001, Y300L, — Y296P, Y296F, Q295K, Q295L, E294N,E294A, or Y300F. or N276Q. or Q295A. E294Q, or E294D. S298D + → Y3001,Y300L, — Y296P, Y296F, Q295K, Q295L, E294N, E294A, or Y300F. or N276Q.or Q295A. E294Q, or E294D. S298P + → Y3001, Y300L, — Y296P, Y296F,Q295K, Q295L, E294N, E294A, or Y300F. or N276Q. or Q295A. E294Q, orE294D. Y296P + → Y3001, Y300L, S298N, S298V, — Q295K, Q295L, E294N,E294A, or Y300F. S298D, S298P, or Q295A. E294Q, or S298A, S298G, E294D.S298T, or S298L. Q295K + → Y3001, Y300L, S298N, S298V, Y296P, Y296F, —E294N, E294A, or Y300F. S298D, S298P, or N276Q. E294Q, or S298A, S298G,E294D. S298T, or S298L. Q295L + → Y3001, Y300L, S298N, S298V, Y296P,Y296F, — E294N, E294A, or Y300F. S298D, S298P, or N276Q. E294Q, orS298A, S298G, E294D. S298T, or S298L. E294N + → Y3001, Y300L, S298N,S298V, Y296P, Y296F, Q295K, Q295L, — or Y300F. S298D, S298P, or N276Q.or Q295A. S298A, S298G, S298T, or S298L. ** Note that table uses EUnumbering as in Kabat.

TABLE 3B Starting Position Position Position Position Position Variant334 333 324 286 276 Y3001 + → K334A, K334R, E33A, E333Q, S324A, S324N,N286Q, N276Q, K334Q, K334N, E333N, S324Q, S324K, N286S, N276A, or K334S,K334E, E333S, or S324E. N286A, or N276K. K334D, K334M, E333K, N286D.K334Y, K334W, E333R, K334H, K334V, or E333D, or K334L. E333G. Y300L + →K334A, K334R, E33A, E333Q, S324A, S324N, N286Q, N276Q, K334Q, K334N,E333N, S324Q, S324K, N286S, N276A, or K334S, K334E, E333S, or S324E.N286A, or N276K. K334D, K334M, E333K, N286D. K334Y, K334W, E333R, K334H,K334V, or E333D, or K334L. E333G. S298N + → K334A, K334R, E33A, E333Q,S324A, S324N, N286Q, N276Q, K334Q, K334N, E333N, S324Q, S324K, N286S,N276A, or K334S, K334E, E333S, or S324E. N286A, or N276K. K334D, K334M,E333K, N286D. K334Y, K334W, E333R, K334H, K334V, or E333D, or K334L.E333G. S298V + → K334A, K334R, E33A, E333Q, S324A, S324N, N286Q, N276Q,K334Q, K334N, E333N, S324Q, S324K, N286S, N276A, or K334S, K334E, E333S,or S324E. N286A, or N276K. K334D, K334M, E333K, N286D. K334Y, K334W,E333R, K334H, K334V, or E333D, or K334L. E333G. S298D + → K334A, K334R,E33A, E333Q, S324A, S324N, N286Q, N276Q, K334Q, K334N, E333N, S324Q,S324K, N286S, N276A, or K334S, K334E, E333S, or S324E. N286A, or N276K.K334D, K334M, E333K, N286D. K334Y, K334W, E333R, K334H, K334V, or E333D,or K334L. E333G. S298P + → K334A, K334R, E33A, E333Q, S324A, S324N,N286Q, N276Q, K334Q, K334N, E333N, S324Q, S324K, N286S, N276A, or K334S,K334E, E333S, or S324E. N286A, or N276K. K334D, K334M, E333K, N286D.K334Y, K334W, E333R, K334H, K334V, or E333D, or K334L. E333G. Y296P + →K334A, K334R, E33A, E333Q, S324A, S324N, N286Q, N276Q, K334Q, K334N,E333N, S324Q, S324K, N286S, N276A, or K334S, K334E, E333S, or S324E.N286A, or N276K. K334D, K334M, E333K, N286D. K334Y, K334W, E333R, K334H,K334V, or E333D, or K334L. E333G. Q295K + → K334A, K334R, E33A, E333Q,S324A, S324N, N286Q, N276Q, K334Q, K334N, E333N, S324Q, S324K, N286S,N276A, or K334S, K334E, E333S, or S324E. N286A, or N276K. K334D, K334M,E333K, N286D. K334Y, K334W, E333R, K334H, K334V, or E333D, or K334L.E333G. Q295L + → K334A, K334R, E33A, E333Q, S324A, S324N, N286Q, N276Q,K334Q, K334N, E333N, S324Q, S324K, N286S, N276A, or K334S, K334E, E333S,or S324E. N286A, or N276K. K334D, K334M, E333K, N286D. K334Y, K334W,E333R, K334H, K334V, or E333D, or K334L. E333G. E294N + → K334A, K334R,E33A, E333Q, S324A, S324N, N286Q, N276Q, K334Q, K334N, E333N, S324Q,S324K, N286S, N276A, or K334S, K334E, E333S, or S324E. N286A, or N276K.K334D, K334M, E333K, N286D. K334Y, K334W, E333R, K334H, K334V, or E333D,or K334L. E333G. ** Note that table uses EU numbering as in Kabat.

In a preferred specific embodiment, the invention encompasses moleculescomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said molecule has a conferred effector function (i.e., in aparticular assay, the modified molecule has an effector functionactivity not detectable in the parent molecule) and/or an alteredaffinity for an FcγR, provided that said variant Fc region does not havea substitution at positions that make a direct contact with FcγR basedon crystallographic and structural analysis of Fc-FcγR interactions,such as those positions disclosed by Sondermann et al., 2000 (Nature,406: 267-273 which is incorporated herein by reference in its entirety).Examples of positions within the Fc region that make a direct contactwith FcγR are amino acids 234-239 (hinge region), amino acids 265-269(B/C loop), amino acids 297-299 (C′/E loop), and amino acids 327-332(F/G) loop. In some embodiments, the molecules of the inventioncomprising variant Fc regions comprise modification of at least oneresidue that makes a direct contact with an FcγR based on structural andcrystallographic analysis.

The FcγR interacting domain maps to the lower hinge region and selectsites within the CH2 and CH3 domains of the IgG heavy chain. Amino acidresidues flanking the actual contact positions and amino acid residuesin the CH3 domain play a role in IgG/FcγR interactions as indicated bymutagenesis studies and studies using small peptide inhibitors,respectively (Sondermann et al., 2000 Nature, 406: 267-273; Diesenhoferet al., 1981, Biochemistry, 20: 2361-2370; Shields et al., 2001, J.Biol. Chem. 276: 6591-6604; each of which is incorporated herein byreference in its entirety). Direct contact as used herein refers tothose amino acids that are within at least 1 Å, at least 2 Å, or atleast 3 Å of each other or within 1 Å, 1.2 Å, 1.5 Å, 1.7 Å or 2 Å VanDer Waals radius. An exemplary list of previously identified sites onthe Fc that affect binding of Fc interacting proteins is listed in theTable 4 below. In some embodiments, the invention encompasses Fcvariants that do not have any modifications at the sites listed below.In other embodiments, the invention encompasses Fc variants comprisingamino acid modifications at one or more sites listed below incombination with other modifications disclosed herein such that suchmodification has a synergistic or additive effect on the property of themutant.

TABLE 4 PREVIOUSLY IDENTIFIED SITES ON THE Fc THAT EFFECT BINDING OF FcINTERACTING PROTEINS. FcR-Fc Domain residue FcRI FcRII FcRIII C1q FcRnCH2 233 C C C C A, B CH2 234 C C C G C A, B CH2 235 C C C G C A, B CH2236 C C C C A, B CH2 237 A, B CH2 238 D A, B CH2 239 C CH2 241 D CH2 243D CH2 246 D CH2 250 E CH2 254 C CH2 255 C CH2 256 C C CH2 258 C B CH2265 C C C F C B CH2 267 C CH2 268 C C B CH2 269 C CH2 270 C C F CH2 272C CH2 276 C CH2 285 C CH2 286 C CH2 288 C CH2 290 C C CH2 292 C CH2 293C CH2 295 C C CH2 296 C B CH2 297 X X X X B CH2 298 B CH2 299 CH2 301 DC C CH2 311 C CH2 312 C CH2 315 C CH2 317 C CH2 322 C C F CH2 326 C F A,B CH2 327 D, C C C A CH2 328 A CH2 329 D, C C C F A CH2 330 CH2 331 C FA CH2 332 CH2 333 C F CH2 334 C CH2 337 C CH2 338 C CH3 339 C CH3 360 CCH3 362 C CH3 376 C CH3 378 C CH3 380 C CH3 382 C CH3 414 C CH3 415 CCH3 424 C CH3 428 E CH3 430 C CH3 433 C CH3 434 C CH3 435 C CH3 436 C

Table 4 lists sites within the Fc region that have previously beenidentified to be important for the Fc-FcR interaction. Columns labeledFcR-Fc identifies the Fc chain contacted by the FcR. Letters identifythe reference in which the data was cited. C is Shields et al., 2001, J.Biol. Chem. 276: 6591-6604; D is Jefferis et al., 1995, Immunol. Lett.44: 111-7; E is Hinton et al; 2004, J. Biol. Chem. 279(8): 6213-6; F isIdusogie et al., 2000, J. Immunol. 164: 4178-4184; each of which isincorporated herein by reference in its entirety.

In another embodiment, the invention encompasses a molecule comprising avariant Fc region, wherein said variant Fc region comprises at least oneamino acid modification relative to a wild-type Fc region, such thatsaid molecule has an enhanced effector function relative to a moleculecomprising a wild-type Fc region, provided that said variant Fc regiondoes not have or is not solely a substitution at any of positions 243,255, 256, 258, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286,289, 290, 292, 293, 294, 295, 296, 298, 300, 301, 303, 305, 307, 309,312, 320, 322, 326, 329, 330, 332, 331, 333, 334, 335, 337, 338, 339,340, 359, 360, 373, 376, 416, 419, 430, 434, 435, 437, 438, 439. In aspecific embodiment, the invention encompasses a molecule comprising avariant Fc region, wherein said variant Fc region comprises at least oneamino acid modification relative to a wild-type Fc region, such thatsaid molecule binds an FcγR with an altered affinity relative to amolecule comprising a wild-type Fc region, provided that said variant Fcregion does not have or is not solely a substitution at any of positions243, 255, 258, 267, 269, 270, 276, 278, 280, 283, 285, 289, 292, 293,294, 295, 296, 300, 303, 305, 307, 309, 320, 322, 329, 332, 331, 337,338, 340, 373, 376, 416, 419, 434, 435, 437, 438, 439 and does not havean alanine at any of positions 256, 290, 298, 312, 326, 333, 334, 359,360, or 430; an asparagine at position 268; a glutamine at position 272;a glutamine, serine, or aspartic acid at position 286; a serine atposition 290; a methionine at position 301; a methionine, glutamine,glutamic acid, or arginine at position 320; a glutamic acid at position322; an asparagine, serine, glutamic acid, or aspartic acid at position326; a lysine at position 330; a glutamine at position 334; a glutamicacid at position 334; a methionine at position 334; a histidine atposition 334; a valine at position 334; a leucine at position 334; aglutamine at position 335; a lysine at position 335; or a threonine atposition 339.

In a specific embodiment, the invention encompasses a moleculecomprising a variant Fc region, wherein said variant Fc region does nothave or is not solely a substitution at any of positions 268, 269, 270,272, 276, 278, 283, 285, 286, 289, 292, 293, 301, 303, 305, 307, 309,320, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 416, 419, 430,434, 435, 437, 438 or 439 and does not have a histidine, glutamine, ortyrosine at position 280; a serine, glycine, threonine or tyrosine atposition 290, an asparagine at position 294, a lysine at position 295; aproline at position 296; a proline, asparagine, aspartic acid, or valineat position 298; or a leucine or isoleucine at position 300. In anotherembodiment, the invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculebinds an FcγR with a reduced affinity relative to molecule comprising awild-type Fc region provided that said variant Fc region does not haveor is not solely a substitution at any of positions 243, 252, 254, 265,268, 269, 270, 278, 289, 292, 293, 294, 295, 296, 298, 300, 301, 303,322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414,416, 419, 434, 435, 437, 438, or 439. In yet another embodiment, theinvention encompasses a molecule comprising a variant Fc region, whereinsaid variant Fc region comprises at least one amino acid modificationrelative to a wild-type Fc region, such that said molecule binds an FcγRwith an enhanced affinity relative to a molecule comprising a wild-typeFc region provided that said variant Fc region does not have or is notsolely a substitution at any of positions 280, 283, 285, 286, 290, 294,295, 298, 300, 301, 305, 307, 309, 312, 315, 331, 333, 334, 337, 340,360, 378, 398, or 430.

In a specific embodiment, the invention encompasses molecule comprisinga variant Fc region, wherein said variant Fc region does not include orare not solely a substitution at any of positions 330, 243, 247, 298,241, 240, 244, 263, 262, 235, 269, or 328 and does not have a leucine atposition 243, an asparagine at position 298, a leucine at position 241,and isoleucine or an alanine at position 240, a histidine at position244, a valine at position 330, or an isoleucine at position 328.

In alternative embodiments, the molecules of the invention, havingvariant Fc regions with enhanced effector function and/or alteredaffinities for activating and/or inhibitory receptors, have one or moreamino acid modifications, wherein said one or more amino acidmodification is a substitution at position 288 with asaparagine, atposition 330 with serine and at position 396 with leucine (MgFc10)(SeeTable 5); or a substitution at position 334 with glutamic acid, atposition 359 with asparagine, and at position 366 with serine (MgFcl3);or a substitution at position 316 with aspartic acid, at position 378with valine, and at position 399 with glutamic acid (MgFc27); or asubstitution at position 247 with leucine, and a substitution atposition 421 with lysine (MgFc31); or a substitution at position 392with threonine, and at position 396 with leucine (MgFc38); or asubstitution at position 221 with glutamic acid, at position 270 withglutamic acid, at position 308 with alanine, at position 311 withhistidine, at position 396 with leucine, and at position 402 withaspartic acid (MgFc42); or a substitution at position 419 withhistidine, and a substitution at position 396 with leucine (MgFc51); ora substitution at position 240 with alanine, and at position 396 withleucine (MgFc52); or a substitution at position 410 with histidine, andat position 396 with leucine (MgFc53); or a substitution at position 243with leucine, at position 305 with isoleucine, at position 378 withaspartic acid, at position 404 with serine, and at position 396 withleucine (MgFc54); or a substitution at position 255 with isoleucine, andat position 396 with leucine (MgFc55); or a substitution at position 370with glutamic acid and at position 396 with leucine (MgFc59); or asubstitution at position 270 with glutamic acid; or a combination of theforegoing. In one specific embodiment, the invention encompasses amolecule comprising a variant Fc region wherein said variant Fc regioncomprises a substitution at position 396 with leucine, at position 270with glutamic acid and at position 243 with leucine. In another specificembodiment the molecule further comprises one or more amino acidmodification such as those disclosed herein.

In some embodiments, the invention encompasses molecules comprising avariant Fc region having an amino acid modification at one or more ofthe following positions: 119, 125, 132, 133, 141, 142, 147, 149, 162,166, 185, 192, 202, 205, 210, 214, 215, 216, 217, 218, 219, 221, 222,223, 224, 225, 227, 229, 231, 232, 233, 235, 240, 241, 242, 243, 244,246, 247, 248, 250, 251, 252, 253, 254, 255, 256, 258, 261, 262, 263,268, 269, 270, 272, 274, 275, 276, 279, 280, 281, 282, 284, 287, 288,289, 290, 291, 292, 293, 295, 298, 301, 303, 304, 305, 306, 307, 308,309, 310, 311, 312, 313, 315, 316, 317, 318, 319, 320, 323, 326, 327,328, 330, 333, 334, 335, 337, 339, 340, 343, 344, 345, 347, 348, 352,353, 354, 355, 358, 359, 360, 361, 362, 365, 366, 367, 369, 370, 371,372, 375, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388,389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 404,406, 407, 408, 409, 410, 411, 412, 414, 415, 416, 417, 419, 420, 421,422, 423, 424, 427, 428, 431, 433, 435, 436, 438, 440, 441, 442, 443,446, or 447. Preferably such mutations result in molecules that havebeen conferred an effector cell mediated function and, optionally, havean altered affinity for an FcγR as determined using methods disclosedand exemplified herein and known to one skilled in the art.

The invention encompasses molecules comprising variant Fc regionsconsisting of or comprising any of the mutations listed in the tablebelow in Table 5.

TABLE 5 EXEMPLARY MUTATIONS SINGLE SITE MUTANTS DOUBLE SITE MUTANTSK392R Q347H, A339V N315I S415I, L251F S132I K290E, L142P P396L G285E,P247H P396H K409R, S166N A162V E334A, K334A R292L R292L. K334E T359NK288N, A330S T366S R255L, E318K V379L F243L, E318K K288N V279L, P395SA330S K246T, Y319F F243L F243I, V379L E318K K288M, K334E V379M K334E,E308D S219Y E233D, K334E V282M K246T, P396H D401V H268D, E318D K222NK246I, K334N K334I K320E, K326E K334E S375C, P396L I377F K288N, K326NP247L P247L, N421K F372Y S298N, W381R K326E R255Q, K326E H224L V284A,F372L F275Y T394M. V397M L398V P247L, E389G K334N K290T, G371D S400PP247L, L398Q S407I P247L, I377F F372Y K326E, G385E T366N S298N, S407RK414N E258D, N384K M352L F241L, E258G T225S K370N, S440N I377N K317N,F423-DELETED K248M P227S, K290E R292G K334E, E380D S298N P291S, P353QD270E V240I, V281M E233G P232S, S304G P247L, L406F D399E, M428L L251F,F372L D399E, G402D D399E, M428L K392T, P396L H268N, P396L K326I, P396LH268D, P396L K210M, P396L L358P, P396L K334N, P396L V379M, P396L P227S,P396L P217S, P396L Q419H, P396L K370E, P396L L242F, P396L R255L, P396LV240A, P396L T250A, P396L P247S, P396L L410H, P396L Q419L, P396L V427A,P396L E258D, P396L N384K, P396L V323I, P396L P244H, P396L V305L, P396LS400F, P396L V303I, P396L A330V, Q419H V263Q, E272D K326E, A330T

In yet other embodiments, the invention encompasses molecules comprisingvariant Fc regions having more than two amino acid modifications. Anon-limiting example of such variants is listed in the table below(Table 6). The invention encompasses mutations listed in Table 6 whichfurther comprise one or more amino acid modifications such as thosedisclosed herein.

TABLE 6 EXEMPLARY COMBINATION VARIANTS D399E, R292L, V185M R301C, M252L,S192T P291S, K288E, H268L, A141V S383N, N384K, T256N, V262L, K218E,R214I, K205E, F149Y, K133M S408I, V215I, V125L G385E, P247H V348M,K334N, F275I, Y202M, K147T H310Y, T289A, Y407V, E258D R292L, P396L,T359N F275I, K334N, V348M F243L. R255L, E318K K334E, T359N, T366S T256S,V305I, K334E, N390S T335N, K370E, A378V, T394M, S424L K334E, T359N,T366S, Q386R K288N, A330S, P396L P244H, L358M, V379M, N384K, V397MP217S, A378V, S408R P247L, I253N, K334N D312E, K327N, I378S D280E,S354F, A431D, L441I K218R, G281D, G385R P247L, A330T, S440G T355N,P387S, H435Q P247L, A431V, S442F P343S, P353L, S375I, S383N E216D,E345K, S375I K288N, A330S, P396L K222N, T335N, K370E, A378V, T394MG316D, A378V, D399E N315I, V379M, T394M K326Q, K334E, T359N, T366SA378V, N390I, V422I V282E, V369I, L406F V397M, T411A, S415N T223I,T256S, L406F L235P, V382M, S304G, V305I, V323I P247L, W313R, E388GD221Y, M252I, A330G, A339T, T359N, V422I, H433L F243I, V379L, G420VA231V, Q386H, V412M T215P, K274N, A287G, K334N, L365V, P396L P244A,K326I, C367R, S375I, K447T R301H, K340E, D399E C229Y, A287T, V379M,P396L, L443V E269K, K290N, Q311R, H433Y E216D, K334R, S375I T335N,P387S, H435Q K246I, Q362H, K370E K334E, E380D, G446V V303I, V369F, M428LK246E, V284M, V308A E293V, Q295E, A327T Y319F, P352L, P396L D221E,D270E, V308A, Q311H, P396L, G402D K290T, N390I, P396L K288R, T307A,K344E, P396L V273I, K326E, L328I, P396L K326I, S408N, P396L K261N,K210M, P396L F243L, V305I, A378D, F404S, P396L K290E, V369A, T393A,P396L K210N, K222I, K320M, P396L P217S, V305I, I309L, N390H, P396LK246N, Q419R, P396L P217A, T359A, P396L V215I, K290V, P396L F275L,Q362H, N384K, P396L A330V, H433Q, V427M V263Q, E272D, Q419H N276Y,T393N, W417R V282L, A330V, H433Y, T436R V284M, S298N, K334E, R355WA330V, G427M, K438R S219T, T225K, D270E, K360R K222E, V263Q, S298NE233G, P247S, L306P S219T, T225K, D270E S254T, A330V, N361D, P243LV284M, S298N, K334E, R355W R416T D270E, G316D, R416G K392T, P396L, D270ER255L, P396L, D270E V240A, P396L, D270E Q419H, P396L, D270E K370E,P396L, D270E P247L, N421K, D270E R292P, V305I R292P, V305I, F243L V284M,R292L, K370N R255L, P396L, D270E, Y300L R255L, P396L, D270E, R292GF243L, D270E, K392N, P396L F243L, R255L, D270E, P296L

In specific embodiments, the variant Fc region has a leucine at position247, a lysine at position 421 and a glutamic acid at position 270(MgFc31/60); a threonine at position 392, a leucine at position 396, aglutamic acid at position 270, and a leucine at position 243(MgFc38/60/F243L); a histidine at position 419, a leucine at position396, and a glutamic acid at position 270 (MGFc51/60); a histidine atposition 419, a leucine at position 396, a glutamic acid at position270, and a leucine at position 243 (MGFc51/60/F243L); an alanine atposition 240, a leucine at position 396, and a glutamic acid at position270 (MGFc52/60); a lysine at position 255 and a leucine at position 396(MgFc55); a lysine at position 255, a leucine at position 396, and aglutamic acid at position 270 (MGFc55/60); a lysine at position 255, aleucine at position 396, a glutamic acid at position 270, and a lysineat position 300 (MGFc55/60/Y300L); a lysine at position 255, a leucineat position 396, a glutamic acid at position 270, and a glycine atposition 292 (MGFc55/60/R292G); a lysine at position 255, a leucine atposition 396, a glutamic acid at position 270, and a leucine at position243 (MgFc55/60/F243L); a glutamic acid at position 370, a leucine atposition 396, and a glutamic acid at position 270 (MGFc59/60); aglutamic acid at position 270, an aspartic acid at position 316, and aglycine at position 416 (MgFc71); a leucine at position 243, a prolineat position 292, an isoleucine at position 305, and a leucine atposition 396 (MGFc74/P396L); a leucine at position 243, a glutamic acidat position 270, an asparagine at position 392 and a leucine at position396; or a leucine at position 243, a leucine at position 255, a glutamicacid at position 270 and a leucine at position 396; a glutamine atposition 297, or any combination of the individual substitutions.

In some embodiments, the molecules of the invention further comprise oneor more glycosylation sites, so that one or more carbohydrate moietiesare covalently attached to the molecule. Preferably, the molecules ofthe invention with one or more glycosylation sites and/or one or moremodifications in the Fc region confer or have an enhanced antibodymediated effector function, e.g., enhanced ADCC activity, compared to aparent antibody. In some embodiments, the invention further comprisesmolecules comprising one or more modifications of amino acids that aredirectly or indirectly known to interact with a carbohydrate moiety ofthe antibody, including but not limited to amino acids at positions 241,243, 244, 245, 245, 249, 256, 258, 260, 262, 264, 265, 296, 299, and301. Amino acids that directly or indirectly interact with acarbohydrate moiety of an antibody are known in the art, see, e.g.,Jefferis et al., 1995 Immunology Letters, 44: 111-7, which isincorporated herein by reference in its entirety.

In another embodiment, the invention encompasses molecules that havebeen modified by introducing one or more glycosylation sites into one ormore sites of the molecules, preferably without altering thefunctionality of the molecules, e.g., binding activity to target antigenor FcγR. Glycosylation sites may be introduced into the variable and/orconstant region of the molecules of the invention. As used herein,“glycosylation sites” include any specific amino acid sequence in anantibody to which an oligosaccharide (i.e., carbohydrates containing twoor more simple sugars linked together) will specifically and covalentlyattach. Oligosaccharide side chains are typically linked to the backboneof an antibody via either N- or O-linkages. N-linked glycosylationrefers to the attachment of an oligosaccharide moiety to the side chainof an asparagine residue. O-linked glycosylation refers to theattachment of an oligosaccharide moiety to a hydroxyamino acid, e.g.,serine, threonine. The molecules of the invention may comprise one ormore glycosylation sites, including N-linked and O-linked glycosylationsites. Any glycosylation site for N-linked or O-linked glycosylationknown in the art may be used in accordance with the instant invention.An exemplary N-linked glycosylation site that is useful in accordancewith the methods of the present invention is the amino acid sequence:Asn-X-Thr/Ser, wherein X may be any amino acid and Thr/Ser indicates athreonine or a serine. Such a site or sites may be introduced into amolecule of the invention using methods well known in the art to whichthis invention pertains. See, for example, “In vitro Mutagenesis,”Recombinant DNA: A Short Course, J. D. Watson, et al. W. H. Freeman andCompany, New York, 1983, chapter 8, pp. 106-116, which is incorporatedherein by reference in its entirety. An exemplary method for introducinga glycosylation site into a molecule of the invention may comprise:modifying or mutating an amino acid sequence of the molecule so that thedesired Asn-X-Thr/Ser sequence is obtained.

In some embodiments, the invention encompasses methods of modifying thecarbohydrate content of a molecule of the invention by adding ordeleting a glycosylation site. Methods for modifying the carbohydratecontent of antibodies are well known in the art and encompassed withinthe invention, see, e.g., U.S. Pat. No. 6,218,149; EP 0 359 096 B1; U.S.Publication No. US 2002/0028486; WO 03/035835; U.S. Publication No.2003/0115614; U.S. Pat. No. 6,218,149; U.S. Pat. No. 6,472,511; all ofwhich are incorporated herein by reference in their entirety. In otherembodiments, the invention encompasses methods of modifying thecarbohydrate content of a molecule of the invention by deleting one ormore endogenous carbohydrate moieties of the molecule. In a specificembodiment, the invention encompasses shifting the glycosylation site ofthe Fc region of an antibody, by modifying positions adjacent to 297. Ina specific embodiment, the invention encompasses modifying position 296so that position 296 and not position 297 is glycosylated.

5.1 Polypeptides and Antibodies with Variant Fc Regions

The present invention is based, in part, on the inventors' discovery ofmethods for engineering the Fc region of an antibody to confer aneffector function activity to the antibody, which the parent antibodydid not exhibit when tested against a target cell. Such methods ofengineering include introducing one or more amino acid modifications(substitutions, deletions or insertions) in one or more portions of theFc region, which modifications introduce a detectable effector functionactivity on the parent antibody. In particular, the modifications alterthe parent antibody's affinity for certain FcγR receptors (e.g.,activating FcγRs, inhibitory FcγRs) and one or more effector functions,such as ADCC. Alternately, the modifications alter the affinity of thevariant Fc region such that the variant Fc regions oligomerize andhomo-oligomers of the modified antibody are formed. The inventors havefound that modification of an Fc region of a chimeric 2B6 or 4D5antibody (anti-FcγRIIB antibody) surprisingly conferred a particulareffector function activity (ADCC) on chimeric 2B6 antibodies, whichnormally exhibit no detectable ADCC activity, and improved effectorfunction activity (particularly ADCC) of chimeric 4D5 antibodies incells with low levels of antigen expression. The inventors have furtherfound that modification of an Fc region of rituximab (anti-CD20monoclonal antibody) conferred effector function activity on therituximab antibody in a patient population whose cells were otherwiserefractory to rituximab-induced effector function activity.

It will be appreciated by one skilled in the art that aside from aminoacid substitutions, the present invention contemplates othermodifications of the Fc region amino acid sequence in order to generatean Fc region variant with one or more altered properties, e.g., enhancedeffector function. The invention contemplates deletion of one or moreamino acid residues of the Fc region in order to, e.g., reduce bindingto an FcγR. Preferably, no more than 5, no more than 10, no more than20, no more than 30, no more than 50 Fc region residues will be deletedaccording to this embodiment of the invention. The Fc region hereincomprising one or more amino acid deletions will preferably retain atleast about 80%, and preferably at least about 90%, and most preferablyat least about 95%, of the wild type Fc region. In some embodiments, oneor more properties of the molecules are maintained such as for example,non-immunogenicity, FcγRIIIA binding, FcγRIIA binding, or a combinationof these properties.

In alternate embodiments, the invention encompasses amino acid insertionto generate the Fc region variants, which variants have alteredproperties including enhanced effector function. In one specificembodiment, the invention encompasses introducing at least one aminoacid residue, for example, one to two amino acid residues and preferablyno more than 10 amino acid residues adjacent to one or more of the Fcregion positions identified herein. In alternate embodiments, theinvention further encompasses introducing at least one amino acidresidue, for example, one to two amino acid residues and preferably nomore than 10 amino acid residues adjacent to one or more of the Fcregion positions known in the art as impacting FcγR interaction and/orbinding.

The invention further encompasses incorporation of unnatural amino acidsto generate the Fc variants of the invention. Such methods are known tothose skilled in the art such as those using the natural biosyntheticmachinery to allow incorporation of unnatural amino acids into proteins,see, e.g., Wang et al., 2002 Chem. Comm. 1:1-11; Wang et al., 2001,Science, 292: 498-500; van Hest et al., 2001. Chem. Comm. 19: 1897-1904,each of which is incorporated herein by reference in its entirety.Alternative strategies focus on the enzymes responsible for thebiosynthesis of amino acyl-tRNA, see, e.g., Tang et al., 2001, J. Am.Chem. 123(44): 11089-11090; Kiick et al., 2001, FEBS Lett. 505(3): 465;each of which is incorporated herein by reference in its entirety.

The effector function properties of the molecules of the invention aredetermined for one or more FcγR mediator effector cell functions asdescribed in Section 5.2.7. The affinities and binding properties of themolecules of the invention for a target antigen or an FcγR are initiallydetermined using in vitro assays (biochemical or immunological basedassays) known in the art for determining antibody-antigen or Fc-FcγRinteractions, i.e., specific binding of an antibody to an antigen or anFc region to an FcγR, respectively, including but not limited to ELISAassay, surface plasmon resonance assay, immunoprecipitation assays (SeeSection 5.2.1). In most preferred embodiments, the molecules of theinvention have similar binding properties in in vivo models (such asthose described and disclosed herein) as those in in vitro based assays.However, the present invention does not exclude molecules of theinvention that do not exhibit the desired phenotype in in vitro basedassays but do exhibit the desired phenotype in vivo. A representativeflow chart of the screening and characterization of molecules of theinvention is described in FIG. 34.

The invention encompasses molecules comprising a variant Fc region thatbinds with a greater affinity to one or more FcγRs. Such moleculespreferably mediate effector function more effectively as discussedinfra. In other embodiments, the invention encompasses moleculescomprising a variant Fc region that bind with a weaker affinity to oneor more FcγRs. In general, increased or added effector function would bedirected to tumor and foreign cells.

The Fc variants of the present invention may be combined with other Fcmodifications, including but not limited to other modifications thatenhance effector function. The invention encompasses combining an Fcvariant of the invention with other Fc modifications to provideadditive, synergistic, or novel properties in antibodies or Fc fusions.Preferably the Fc variants of the invention enhance the phenotype of themodification with which they are combined. For example, if an Fc variantof the invention is combined with a mutant known to bind FcγRIIIA with ahigher affinity than a comparable molecule comprising a wild type Fcregion; the combination with a mutant of the invention results in agreater fold enhancement in FcγRIIIA affinity.

In one embodiment, the Fc variants of the present invention may becombined with other known Fc variants such as those disclosed in Duncanet al, 1988, Nature 332:563-564; Lund et al., 1991, J. Immunol.147:2657-2662; Lund et al, 1992, Mol Immunol 29:53-59; Alegre et al,1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl.Acad Sci USA 92:11980-11984; Jefferis et al, 1995, Immunol Lett.44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et al, 1996,Immunol Lett 54:101-104; Lund et al, 1996, J Immunol 157:49634969;Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogie et al, 2000, JImmunol 164:41784184; Reddy et al, 2000, J Immunol 164:1925-1933; Xu etal., 2000, Cell Immunol 200:16-26; Idusogie et al, 2001, J Immunol166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferiset al, 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem SocTrans 30:487-490); U.S. Pat. No. 5,624,821; U.S. Pat. No. 5,885,573;U.S. Pat. No. 6,194,551; PCT WO 00/42072; PCT WO 99/58572; each of whichis incorporated herein by reference in its entirety.

In some embodiments, the Fc variants of the present invention areincorporated into an antibody or Fc fusion that comprises one or moreengineered glycoforms, i.e., a carbohydrate composition that iscovalently attached to an antibody comprising an Fc region, wherein saidcarbohydrate composition differs chemically from that of a parentantibody comprising an Fc region. Engineered glycoforms may be usefulfor a variety of purposes, including, but not limited to, enhancingeffector function. Engineered glycoforms may be generated by any methodknown to one skilled in the art, for example by using engineered orvariant expression strains, by co-expression with one or more enzymes,for example, DI N-acetylglucosaminyltransferase III (GnTI11), byexpressing an antibody comprising an Fc region in various organisms orcell lines from various organisms, or by modifying carbohydrate(s) afterthe antibody comprising Fc region has been expressed. Methods forgenerating engineered glycoforms are known in the art, and include butare not limited to those described in Umana et al, 1999, Nat. Biotechnol17:176-180; Davies et al., 2001 Biotechnol Bioeng 74:288-294; Shields etal, 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J BiolChem 278:3466-3473) U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370;U.S. Ser. No. 10/113,929; PCT WO 00/61739A1; PCT WO 01/292246A1; PCT WO02/311140A1; PCT WO 02/30954A1; Potillegent™ technology (Biowa, Inc.Princeton, N.J.); GlycoMAb™ glycosylation engineering technology(GLYCART biotechnology AG, Zurich, Switzerland); each of which isincorporated herein by reference in its entirety. See, e.g., WO00061739; EA01229125; US 20030115614; Okazaki et al., 2004, JMB, 336:1239-49 each of which is incorporated herein by reference in itsentirety.

The Fc variants of the present invention may be optimized for a varietyof properties. Properties that may be optimized include, but are notlimited to, conferred or enhanced effector function, enhanced or reducedaffinity for an FcγR, or conferred oligomerization activity. In apreferred embodiment, the Fc variants of the present invention areoptimized to possess enhanced affinity for a human activating FcγR,preferably FcγR, FcγRIIA, FcγRIIc, FcγRIIIA, and FcγRIIIB, mostpreferably FcγRIIIA. In an alternate preferred embodiment, the Fcvariants are optimized to possess reduced affinity for the humaninhibitory receptor FcγRIIB. These preferred embodiments are anticipatedto provide antibodies and Fc fusions with new or enhanced therapeuticproperties in humans, for example, enhanced effector function andgreater anti-cancer potency as described and exemplified herein. Thesepreferred embodiments are anticipated to provide antibodies and Fcfusions with enhanced tumor elimination in mouse xenograft tumor models.

In an alternate embodiment the Fc variants of the present invention areoptimized to have reduced affinity for a human FcγR, including but notlimited to FcγRI, FcγRIIA, FcγRIIB, FcγRIIc, FcγRIIIA, and FcγRIIIB.These embodiments are anticipated to provide antibodies and Fc fusionswith enhanced therapeutic properties in humans, for example, reducedtoxicity.

In alternate embodiments, the Fc variants of the present inventionpossess a conferred effector function and/or enhanced or reducedaffinity for FcγRs from non-human organisms, including, but not limitedto, mice, rats, rabbits, and monkeys. Fc variants that are optimized foreffector function in a non-human or binding to a non-human FcγR may finduse in experimentation. For example, mouse models are available for avariety of diseases that enable testing of properties such as efficacy,toxicity, and pharmacokinetics for a given drug candidate. As is knownin the art, cancer cells can be grafted or injected into mice to mimic ahuman cancer, a process referred to as xenografting. Testing ofantibodies or Fc fusions that comprise Fc variants that confer aneffector function and/or are optimized for one or more mouse FcγRs, mayprovide valuable information with regard to the efficacy of the antibodyor Fc fusion, its mechanism of action, and the like.

In certain embodiments, while it is preferred to alter binding to anFcγR, the instant invention further contemplates Fc variants withaltered binding affinity to the neonatal receptor (FcRn). Although notintending to be bound by a particular mechanism of action, Fc regionvariants with improved affinity for FcRn are anticipated to have longerserum half-lives, and such antibodies will have useful applications inmethods of treating mammals where long half-life of the administeredpolypeptide is desired, e.g., to treat a chronic disease or disorder.Although not intending to be bound by a particular mechanism of action,Fc region variants with decreased FcRn binding affinity, on thecontrary, are expected to have shorter half-lives, and such antibodiesmay, for example, be administered to a mammal where a shortenedcirculation time may be advantageous, e.g., for in vivo diagnosticimaging or for polypeptides which have toxic side effects when leftcirculating in the blood stream for extended periods. Fc region variantswith decreased FcRn binding affinity are anticipated to be less likelyto cross the placenta, and thus may be utilized in the treatment ofdiseases or disorders in pregnant women.

In other embodiments, these variants may be combined with other known Fcmodifications with altered FcRn affinity such as those disclosed inInternational Publication Nos. WO 98/23289; and WO 97/34631; and U.S.Pat. No. 6,277,375, each of which is incorporated herein by reference inits entirety.

The invention encompasses any other method known in the art forgenerating molecules, e.g., antibodies, having an increased half-life invivo, for example, by introducing one or more amino acid modifications(i.e., substitutions, insertions or deletions) into an IgG constantdomain, or FcRn binding fragment thereof (preferably a Fc or hinge-Fcdomain fragment). See, e.g., International Publication Nos. WO 98/23289;and WO 97/34631; and U.S. Pat. No. 6,277,375, each of which isincorporated herein by reference in its entirety to be used incombination with the Fc variants of the invention. Further, molecules,e.g., antibodies, of the invention can be conjugated to albumin in orderto make the antibody or antibody fragment more stable in vivo or have alonger half-life in vivo. The techniques well-known in the art, see,e.g., International Publication Nos. WO 93/15199, WO 93/15200, and WO01/77137, and European Patent No. EP 413,622, all of which areincorporated herein by reference in their entirety.

The variant(s) described herein may be subjected to furthermodifications, often times depending on the intended use of the variant.Such modifications may involve further alteration of the amino acidsequence (substitution, insertion and/or deletion of amino acidresidues), fusion to heterologous polypeptide(s) and/or covalentmodifications. Such further modifications may be made prior to,simultaneously with, or following, the amino acid modification(s)disclosed herein which results in altered properties such as an enhancedbinding to target antigen, conferred oligomerization activity, orenhanced effector function and/or alteration of Fc receptor binding.

Alternatively or additionally, the invention encompasses combining theamino acid modifications disclosed herein with one or more further aminoacid modifications that confer or enhance additional effector functions,e.g., C1q binding and/or complement dependent cytoxicity function, ofthe Fc region as determined in vitro and/or in vivo. The further aminoacid substitutions described herein will generally serve to confer theactivity on a parent antibody that does not exhibit detectable levels ofthe activity or enhance the ability of the starting antibody to bind toC1q and/or complement dependent cytotoxicity (CDC) function. Forexample, the starting antibody may be unable to bind C1q and/or mediateCDC and may be modified according to the teachings herein such that itacquires these further effector functions. Moreover, antibodies withpreexisting C1q binding activity, optionally further having the abilityto mediate CDC may be modified such that one or both of these activitiesare enhanced. In some embodiments, the invention encompasses variant Fcregions with altered CDC activity without any alteration in C1q binding.In yet other embodiments, the invention encompasses variant Fc regionswith altered CDC activity and altered C1q binding.

To generate an Fc region with altered C1q binding and/or complementdependent cytotoxicity (CDC) function, the amino acid positions to bemodified are generally selected from positions 270, 322, 326, 327, 329,331, 333, and 334, where the numbering of the residues in an IgG heavychain is that of the EU index as in Kabat et al., Sequences of Proteinsof Immunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (199). These amino acidmodifications may be combined with one or more Fc modificationsdisclosed herein to provide a synergistic or additive effect on C1qbinding and/or CDC activity. In other embodiments, the inventionencompasses Fc variants with altered C1q binding and/or complementdependent cytotoxicity (CDC) function comprising an amino acidsubstitution at position 396 with leucine and at position 255 withleucine; or an amino acid substitution at position 396 with leucine andat position 419 with histidine; an amino acid substitution at position396 with leucine and at position 370 with glutamic acid; an amino acidsubstitution at position 396 with leucine and at position 240 withalanine; an amino acid substitution at position 396 with leucine and atposition 392 with threonine; an amino acid substitution at position 247with leucine and at position 421 with lysine. The invention encompassesany known modification of the Fc region which alters C1q binding and/orcomplement dependent cytotoxicity (CDC) function such as those disclosedin Idusogie et al., 2001, J. Immunol. 166(4) 2571-5; Idusogie et al., J.Immunol. 2000 164(8): 4178-4184; each of which is incorporated herein byreference in its entirety.

As disclosed above, the invention encompasses an Fc region with alteredeffector function, e.g., modified C1q binding and/or FcR binding andthereby altered CDC activity and/or ADCC activity. In specificembodiments, the invention encompasses variant Fc regions with improvedC1q binding and improved FcγRIII binding; e.g. having both improved ADCCactivity and improved CDC activity. In alternative embodiments, theinvention encompasses a variant Fc region with reduced CDC activityand/or reduced ADCC activity. In other embodiments, one may increaseonly one of these activities, and optionally also reduce the otheractivity, e.g. to generate an Fc region variant with improved ADCCactivity, but reduced CDC activity and vice versa.

A. Mutants with Enhanced Altered Affinities for FcγRIIIA and/or FcγRIIA

The invention encompasses molecules comprising a variant Fc region,having one or more amino acid modifications (e.g., substitutions) in oneor more regions, wherein such modifications alter the affinity of thevariant Fc region for an activating FcγR. In some embodiments, moleculesof the invention comprise a variant Fc region, having one or more aminoacid modifications (e.g., substitutions) in one or more regions, whichmodifications increase the affinity of the variant Fc region forFcγRIIIA and/or FcγRIIA by at least 2-fold, relative to a comparablemolecule comprising a wild-type Fc region. In another specificembodiment, molecules of the invention comprise a variant Fc region,having one or more amino acid modifications (e.g., substitutions) in oneor more regions, which modifications increase the affinity of thevariant Fc region for FcγRIIIA and/or FcγRIIA by greater than 2 fold,relative to a comparable molecule comprising a wild-type Fc region. Inother embodiments of the invention, the one or more amino acidmodifications increase the affinity of the variant Fc region forFcγRIIIA and/or FcγRIIA by at least 3-fold, 4-fold, 5-fold, 6-fold,8-fold, or 10-fold relative to a comparable molecule comprising awild-type Fc region. In yet other embodiments of the invention the oneor more amino acid modifications decrease the affinity of the variant Fcregion for FcγRIIIA and/or FcγRIIA by at least 3-fold, 4-fold, 5-fold,6-fold, 8-fold, or 10-fold relative to a comparable molecule comprisinga wild-type Fc region. Such fold increases are preferably determined byan ELISA or surface plasmon resonance assays. In a specific embodiment,the one or more amino acid modifications do not include or are notsolely a substitution at any one of positions 329, 331, or 322 with anyamino acid. In certain embodiments, the one or more amino acidmodifications do not include or are not solely a substitution with anyone of alanine at positions 243, 256, 290, 298, 312, 333, 334, 359, 360,or 430; with lysine at position 330; with threonine at position 339;with methionine or arginine at position 320; with serine, asparagine,aspartic acid, or glutamic acid at position 326 with glutamine, glutamicacid, methionine, histidine, valine, or leucine at position 334. Inanother specific embodiment, the one or more amino acid modifications donot include or are not solely a substitution at any of positions 280,290, 300, 294, or 295. In another more specific embodiment, the one ormore amino acid modifications do not include or are not solely asubstitution at position 300 with leucine or isoleucine; at position 295with lysine; at position 294 with asparagine; at position 298 withvaline; aspartic acid proline, asparagine, or valine; at position 280with histidine, glutamine or tyrosine; at position 290 with serine,glycine, threonine or tyrosine.

In another specific embodiment, the invention encompasses a moleculecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said polypeptide specifically binds FcγRIIA with a greateraffinity than a comparable molecule comprising the wild-type Fc regionbinds FcγRIIA, provided that said variant Fc region does not have analanine at any of positions 256, 290, 326, 255, 258, 267, 272, 276, 280,283, 285, 286, 331, 337, 268, 272, or 430; an asparagine at position268; a glutamine at position 272; a glutamine, serine, or aspartic acidat position 286; a serine at position 290; a methionine, glutamine,glutamic acid, or arginine at position 320; a glutamic acid at position322; a serine, glutamic acid, or aspartic acid at position 326; a lysineat position 330; a glutamine at position 335; or a methionine atposition 301. In a specific embodiment, molecules of the inventioncomprise a variant Fc region, having one or more amino acidmodifications (e.g., substitutions) in one or more regions, whichmodifications increase the affinity of the variant Fc region for FcγRIIAby at least 2-fold, relative to a comparable molecule comprising awild-type Fc region. In another specific embodiment, molecules of theinvention comprise a variant Fc region, having one or more amino acidmodifications (e.g., substitutions) in one or more regions, whichmodifications increase the affinity of the variant Fc region for FcγRIIAby greater than 2 fold, relative to a comparable molecule comprising awild-type Fc region. In other embodiments of the invention the one ormore amino acid modifications increase the affinity of the variant Fcregion for FcγRIIA by at least 3-fold, 4-fold, 5-fold, 6-fold, 8-fold,or 10-fold relative to a comparable molecule comprising a wild-type Fcregion.

In a specific embodiment, the invention encompasses molecules comprisinga variant Fc region, having one or more amino acid modifications (e.g.,substitutions but also include insertions or deletions), whichmodifications increase the affinity of the variant Fc region forFcγRIIIA and/or FcγRIIA by at least 65%, at least 70%, at least 75%, atleast 85%, at least 90%, at least 95%, at least 99%, at least 100%, atleast 150%, and at least 200%, relative to a comparable moleculecomprising a wild-type Fc region.

In a specific embodiment, the one or more amino acid modifications whichincrease the affinity of the variant Fc region comprise a substitutionat position 347 with histidine, and at position 339 with valine; or asubstitution at position 425 with isoleucine and at position 215 withphenylalanine; or a substitution at position 408 with isoleucine, atposition 215 with isoleucine, and at position 125 with leucine; or asubstitution at position 385 with glutamic acid and at position 247 withhistidine; or a substitution at position 348 with methionine, atposition 334 with asparagine, at position 275 with isoleucine, atposition 202 with methionine, and at position 147 with threonine; or asubstitution at position 275 with isoleucine, at position 334 withasparagine, and at position 348 with methionine; or a substitution atposition 279 with leucine and at position 395 with serine; or asubstitution at position 246 with threonine and at position 319 withphenylalanine; or a substitution at position 243 with isoleucine and atposition 379 with leucine; or a substitution at position 243 withleucine, at position 255 with leucine and at position 318 with lysine;or a substitution at position 334 with glutamic acid, at position 359with asparagine, and at position 366 with serine; or a substitution atposition 288 with methionine and at position 334 with glutamic acid; ora substitution at position 334 with glutamic acid and at position 380with aspartic acid; or a substitution at position 256 with serine, atposition 305 with isoleucine, at position 334 with glutamic acid and atposition 390 with serine; or a substitution at position 335 withasparagine, at position 370 with glutamic acid, at position 378 withvaline, at position 394 with methionine, and at position 424 withleucine; or a substitution at position 233 with aspartic acid and atposition 334 with glutamic acid; or a substitution at position 334 withglutamic acid, at position 359 with asparagine, at position 366 withserine, and at position 386 with arginine; or a substitution at position246 with threonine and at position 396 with histidine; or a substitutionat position 268 with aspartic acid and at position 318 with asparticacid; or a substitution at position 288 with asparagine, at position 330with serine, and at position 396 with leucine; or a substitution atposition 244 with histidine, at position 358 with methionine, atposition 379 with methionine, at position 384 with lysine and atposition 397 with methionine; or a substitution at position 217 withserine, at position 378 with valine, and at position 408 with arginine;or a substitution at position 247 with leucine, at position 253 withasparagine, and at position 334 with asparagine; or a substitution atposition 246 with isoleucine, and at position 334 with asparagine; or asubstitution at position 320 with glutamic acid and at position 326 withglutamic acid; or a substitution at position 375 with cysteine and atposition 396 with leucine; or a substitution at position 243 withleucine, at position 270 with glutamic acid, at position 392 withasparagine and at position 396 with leucine; or a substitution atposition 243 with leucine, at position 255 with leucine, at position 270with glutamic acid and at position 396 with leucine; or a substitutionat position 300 with leucine. Examples of other amino acid substitutionsthat results in an enhanced affinity for FcγRIIIA in vitro are disclosedbelow and summarized in Table 5.

The invention encompasses a molecule comprising a variant Fc region,wherein said variant Fc region comprises a substitution at position 243with isoleucine and at position 379 with leucine, such that saidmolecule binds FcγRIIIA with about a 1.5 fold higher affinity than acomparable molecule comprising the wild type Fc region binds FcγRIIIA,as determined by an ELISA assay. In a specific embodiment, the inventionencompasses a molecule comprising a variant Fc region, wherein saidvariant Fc region comprises a substitution at position 288 withasparagine, at position 330 with serine, and at position 396 withleucine, such that said molecule binds FcγRIIIA with about a 5 foldhigher affinity than a comparable molecule comprising the wild type Fcregion binds FcγRIIIA, as determined by an ELISA assay. In a specificembodiment, the invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises a substitution atposition 243 with leucine and at position 255 with leucine such thatsaid molecule binds FcγRIIIA with about a 1 fold higher affinity than acomparable molecule comprising the wild type Fc region binds FcγRIIIA,as determined by an ELISA assay. In a specific embodiment, the inventionencompasses a molecule comprising a variant Fc region, wherein saidvariant Fc region comprises a substitution at position 334 with glutamicacid, at position 359 with asparagine, and at position 366 with serine,such that said molecule binds FcγRIIIA with about a 1.5 fold higheraffinity than a comparable molecule comprising the wild type Fc regionbinds FcγRIIIA, as determined by an ELISA assay. In a specificembodiment, the invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises a substitution atposition 288 with methionine and at position 334 with glutamic acid,such that said molecule binds FcγRIIIA with about a 3 fold higheraffinity than a comparable molecule comprising the wild type Fc regionbinds FcγRIIIA, as determined by an ELISA assay. In a specificembodiment, the invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises a substitution atposition 316 with aspartic acid, at position 378 with valine, and atposition 399 with glutamic acid, such that said molecule binds FcγRIIIAwith about a 1.5 fold higher affinity than a comparable moleculecomprising the wild type Fc region binds FcγRIIIA, as determined by anELISA assay. In a specific embodiment, the invention encompasses amolecule comprising a variant Fc region, wherein said variant Fc regioncomprises a substitution at position 315 with isoleucine, at position379 with methionine, and at position 399 with glutamic acid, such thatsaid molecule binds FcγRIIIA with about a 1 fold higher affinity than acomparable molecule comprising the wild type Fc region binds FcγRIIIA,as determined by an ELISA assay. In a specific embodiment, the inventionencompasses a molecule comprising a variant Fc region, wherein saidvariant Fc region comprises a substitution at position 243 withisoleucine, at position 379 with leucine, and at position 420 withvaline, such that said molecule binds FcγRIIIA with about a 2.5 foldhigher affinity than a comparable molecule comprising the wild type Fcregion binds FcγRIIIA, as determined by an ELISA assay. In a specificembodiment, the invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises a substitution atposition 247 with leucine, and at position 421 with lysine, such thatsaid molecule binds FcγRIIIA with about a 3 fold higher affinity than acomparable molecule comprising the wild type Fc region binds FcγRIIIA,as determined by an ELISA assay. In a specific embodiment, the inventionencompasses a molecule comprising a variant Fc region, wherein saidvariant Fc region comprises a substitution at position 392 withthreonine and at position 396 with leucine such that said molecule bindsFcγRIIIA with about a 4.5 fold higher affinity than a comparablemolecule comprising the wild type Fc region binds FcγRIIIA, asdetermined by an ELISA assay. In a specific embodiment, the inventionencompasses a molecule comprising a variant Fc region, wherein saidvariant Fc region comprises a substitution at position 293 with valine,at position 295 with glutamic acid, and at position 327 with threonine,such that said molecule binds FcγRIIIA with about a 1.5 fold higheraffinity than a comparable molecule comprising the wild type Fc regionbinds FcγRIIIA, as determined by an ELISA assay. In a specificembodiment, the invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises a substitution atposition 268 with asparagine and at position 396 with leucine, such thatsaid molecule binds FcγRIIIA with about a 2 fold higher affinity than acomparable molecule comprising the wild type Fc region binds FcγRIIIA,as determined by an ELISA assay. In a specific embodiment, the inventionencompasses a molecule comprising a variant Fc region, wherein saidvariant Fc region comprises a substitution at position 319 withphenylalanine, at position 352 with leucine, and at position 396 withleucine, such that said molecule binds FcγRIIIA with about a 2 foldhigher affinity than a comparable molecule comprising the wild type Fcregion binds FcγRIIIA, as determined by an ELISA assay.

In a specific embodiment, the invention encompasses a moleculecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said molecule specifically binds FcγRIIIA with a greateraffinity than a comparable molecule comprising the wild-type Fc region,wherein said at least one amino acid modification comprises substitutionat position 396 with histidine. In a specific embodiment, the inventionencompasses a molecule comprising a variant Fc region, wherein saidvariant Fc region comprises at least one amino acid modificationrelative to a wild-type Fc region, such that said molecule specificallybinds FcγRIIIA with a greater affinity than a comparable moleculecomprising the wild-type Fc region, wherein said at least one amino acidmodification comprises substitution at position 248 with methionine. Theinvention encompasses a molecule comprising a variant Fc region, whereinsaid variant Fc region comprises at least one amino acid modificationrelative to a wild-type Fc region, such that said polypeptidespecifically binds FcγRIIIA with a similar affinity than a comparablemolecule comprising the wild-type Fc region, wherein said at least oneamino acid modification comprises substitution at position 392 witharginine. The invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculespecifically binds FcγRIIIA with a similar affinity than a comparablemolecule comprising the wild-type Fc region, wherein said at least oneamino acid modification comprises substitution at position 315 withisoleucine. The invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculespecifically binds FcγRIIIA with a similar affinity than a comparablemolecule comprising the wild-type Fc region, wherein said at least oneamino acid modification comprises substitution at position 132 withisoleucine. The invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculespecifically binds FcγRIIIA with a similar affinity than a comparablemolecule comprising the wild-type Fc region, wherein said at least oneamino acid modification comprises substitution at position 162 withvaline. The invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculespecifically binds FcγRIIIA with a greater affinity than a comparablemolecule comprising the wild-type Fc region, wherein said at least oneamino acid modification comprises substitution at position 396 withleucine. The invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculespecifically binds FcγRIIIA with a greater affinity than a comparablepolypeptide comprising the wild-type Fc region, wherein said at leastone amino acid modification comprises substitution at position 379 withmethionine. The invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculespecifically binds FcγRIIIA with a greater affinity than a comparablemolecule comprising the wild-type Fc region, wherein said at least oneamino acid modification comprises substitution at position 219 withtyrosine. The invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculespecifically binds FcγRIIIA with a greater affinity than a comparablemolecule comprising the wild-type Fc region, wherein said at least oneamino acid modification comprises substitution at position 282 withmethionine. The invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculespecifically binds FcγRIIIA with a greater affinity than a comparablemolecule comprising the wild-type Fc region, wherein said at least oneamino acid modification comprises substitution at position 401 withvaline. The invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculespecifically binds FcγRIIIA with a greater affinity than a comparablemolecule comprising the wild-type Fc region, wherein said at least oneamino acid modification comprises substitution at position 222 withasparagine. The invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculespecifically binds FcγRIIIA with a greater affinity than a comparablemolecule comprising the wild-type Fc region, wherein said at least oneamino acid modification comprises substitution at position 334 withglutamic acid. The invention encompasses a molecule comprising a variantFc region, wherein said variant Fc region comprises at least one aminoacid modification relative to a wild-type Fc region, such that saidmolecule specifically binds FcγRIIIA with a greater affinity than acomparable molecule comprising the wild-type Fc region, wherein said atleast one amino acid modification comprises substitution at position 377with phenylalanine. The invention encompasses a molecule comprising avariant Fc region, wherein said variant Fc region comprises at least oneamino acid modification relative to a wild-type Fc region, such thatsaid molecule specifically binds FcγRIIIA with a greater affinity than acomparable molecule comprising the wild-type Fc region, wherein said atleast one amino acid modification comprises substitution at position 334with isoleucine. The invention encompasses a molecule comprising avariant Fc region, wherein said variant Fc region comprises at least oneamino acid modification relative to a wild-type Fc region, such thatsaid molecule specifically binds FcγRIIIA with a greater affinity than acomparable molecule comprising the wild-type Fc region, wherein said atleast one amino acid modification comprises substitution at position 247with leucine. The invention encompasses a molecule comprising a variantFc region, wherein said variant Fc region comprises at least one aminoacid modification relative to a wild-type Fc region, such that saidmolecule specifically binds FcγRIIIA with a greater affinity than acomparable molecule comprising the wild-type Fc region, wherein said atleast one amino acid modification comprises substitution at position 326with glutamic acid. The invention encompasses a molecule comprising avariant Fc region, wherein said variant Fc region comprises at least oneamino acid modification relative to a wild-type Fc region, such thatsaid molecule specifically binds FcγRIIIA with a greater affinity than acomparable molecule comprising the wild-type Fc region, wherein said atleast one amino acid modification comprises substitution at position 372with tyrosine. The invention encompasses a molecule comprising a variantFc region, wherein said variant Fc region comprises at least one aminoacid modification relative to a wild-type Fc region, such that saidmolecule specifically binds FcγRIIIA with a greater affinity than acomparable molecule comprising the wild-type Fc region, wherein said atleast one amino acid modification comprises substitution at position 224with leucine.

The invention encompasses a molecule comprising a variant Fc region,wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculespecifically binds FcγRIIIA with a greater affinity than a comparablemolecule comprising the wild-type Fc region, wherein said at least oneamino acid modification comprises substitution at position 275 withtyrosine. The invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculespecifically binds FcγRIIIA with a greater affinity than a comparablemolecule comprising the wild-type Fc region, wherein said at least oneamino acid modification comprises substitution at position 398 withvaline. The invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculespecifically binds FcγRIIIA with a greater affinity than a comparablemolecule comprising the wild-type Fc region, wherein said at least oneamino acid modification comprises substitution at position 334 withasparagine. The invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculespecifically binds FcγRIIIA with a greater affinity than a comparablemolecule comprising the wild-type Fc region, wherein said at least oneamino acid modification comprises substitution at position 400 withproline. The invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculespecifically binds FcγRIIIA with a greater affinity than a comparablemolecule comprising the wild-type Fc region, wherein said at least oneamino acid modification comprises substitution at position 407 withisoleucine. The invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculespecifically binds FcγRIIIA with a greater affinity than a comparablemolecule comprising the wild-type Fc region, wherein said at least oneamino acid modification comprises substitution at position 372 withtyrosine. The invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculespecifically binds FcγRIIIA with a similar affinity than a comparablemolecule comprising the wild-type Fc region, wherein said at least oneamino acid modification comprises substitution at position 366 withasparagine. The invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculespecifically binds FcγRIIIA with a reduced affinity than a comparablemolecule comprising the wild-type Fc region, wherein said at least oneamino acid modification comprises substitution at position 414 withasparagine. The invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculespecifically binds FcγRIIIA with a reduced affinity than a comparablemolecule comprising the wild-type Fc region, wherein said at least oneamino acid modification comprises substitution at position 225 withserine. The invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculespecifically binds FcγRIIIA with a reduced affinity than a comparablemolecule comprising the wild-type Fc region, wherein said at least oneamino acid modification comprises substitution at position 377 withasparagine.

In a specific embodiment, the invention encompasses a moleculecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said molecule specifically binds FcγRIIIA with about a 2 foldgreater affinity than a comparable molecule comprising the wild-type Fcregion as determined by an ELISA assay, wherein said at least one aminoacid modification comprises substitution at position 379 withmethionine. In another specific embodiment, the invention encompasses amolecule comprising a variant Fc region, wherein said variant Fc regioncomprises at least one amino acid modification relative to a wild-typeFc region, such that said molecule specifically binds FcγRIIIA withabout a 1.5 fold greater affinity than a comparable molecule comprisingthe wild-type Fc region as determined by an ELISA assay, wherein said atleast one amino acid modification comprises substitution at position 248with methionine.

In some embodiments, the molecules of the invention have an alteredaffinity for FcγRIIIA and/or FcγRIIA as determined using in vitro assays(biochemical or immunological based assays) known in the art fordetermining Fc-FcγR interactions, i.e., specific binding of an Fc regionto an FcγR including but not limited to ELISA assay, surface plasmonresonance assay, immunoprecipitation assays (See Section 5.2.1).Preferably, the binding properties of these molecules with alteredaffinities for activating FcγR receptors are also correlated to theiractivity as determined by in vitro functional assays for determining oneor more FcγR mediator effector cell functions, e.g., molecules withvariant Fc regions with enhanced affinity for FcγRIIIA have a conferredor an enhanced ADCC activity. In most preferred embodiments, themolecules of the invention that have an altered binding property for anactivating Fc receptor, e.g., FcγRIIIA in an in vitro assay, also havean altered binding property in in vivo models (such as those describedand disclosed herein). However, the present invention does not excludemolecules of the invention that do not exhibit an altered FcγR bindingin in vitro based assays but do exhibit the desired phenotype in vivo.

B. Mutants with Enhanced Affinity for FcγRIIIA and Reduced or NoAffinity for FcγRIIB

In a specific embodiment, the molecules of the invention comprise avariant Fc region, having one or more amino acid modifications (i.e.,substitutions) in one or more regions, which one or more modificationsincrease the affinity of the variant Fc region for FcγRIIIA and decreasethe affinity of the variant Fc region for FcγRIIB, relative to acomparable molecule comprising a wild-type Fc region which bindsFcγRIIIA and FcγRIIB with wild-type affinity. In a certain embodiment,the one or more amino acid modifications do not include or are notsolely a substitution with alanine at any of positions 256, 298, 333,334, 280, 290, 294, 298, or 296; or a substitution at position 298 withasparagine, valine, aspartic acid, or proline; or a substitution 290with serine. In certain amino embodiments, the one or more amino acidmodifications increases the affinity of the variant Fc region forFcγRIIIA by at least 65%, at least 70%, at least 75%, at least 85%, atleast 90%, at least 95%, at least 99%, at least 100%, at least 200%, atleast 300%, at least 400% and decreases the affinity of the variant Fcregion for FcγRIIB by at least 65%, at least 70%, at least 75%, at least85%, at least 90%, at least 95%, at least 99%, at least 100%, at least200%, at least 300%, at least 400%.

In a specific embodiment, the molecule of the invention comprising avariant Fc region with an enhanced affinity for FcγRIIIA and a loweredaffinity or no affinity for FcγRIIB, as determined based on an ELISAassay and/or an ADCC based assay using ch-4-4-20 antibody carrying thevariant Fc region, comprises a substitution at any of the following: atposition 275 with isoleucine, at position 334 with asparagine, and atposition 348 with methionine; or a substitution at position 279 withleucine and at position 395 with serine; or a substitution at position246 with threonine and at position 319 with phenylalanine; or asubstitution at position 243 with leucine, at position 255 with leucine,and at position 318 with lysine; or a substitution at position 334 withglutamic acid, at position 359 with asparagine and at position 366 withserine; or a substitution at position 334 with glutamic acid and atposition 380 with aspartic acid; or a substitution at position 256 withserine, at position 305 with isoleucine, at position 334 with glutamicacid, and at position 390 with serine; or a substitution at position 335with asparagine, at position 370 with glutamic acid, at position 378with valine, at position 394 with methionine and at position 424 withleucine; or a substitution at position 233 with aspartic acid and atposition 334 with glutamic acid; or a substitution at position 334 withglutamic acid, at position 359 with asparagine, at position 366 withserine and at position 386 with arginine; or a substitution at position312 with glutamic acid, at position 327 with asparagine, and at position378 with serine; or a substitution at position 288 with asparagine andat position 326 with asparagine; or a substitution at position 247 withleucine and at position 421 with lysine; or a substitution at position298 with asparagine and at position 381 with arginine; or a substitutionat position 280 with glutamic acid, at position 354 with phenylalanine,at position 431 with aspartic acid, and at position 441 with isoleucine;or a substitution at position 255 with glutamine and at position 326with glutamic acid; or a substitution at position 218 with arginine, atposition 281 with aspartic acid and at position 385 with arginine; or asubstitution at position 247 with leucine, at position 330 withthreonine and at position 440 with glycine; or a substitution atposition 284 with alanine and at position 372 with leucine; or asubstitution at position 335 with asparagine, as position 387 withserine and at position 435 with glutamine; or a substitution at position247 with leucine, at position 431 with valine and at position 442 withphenylalanine.

In a specific embodiment, the molecule of the invention comprising avariant Fc region with an enhanced affinity for FcγRIIIA and a loweredaffinity or no affinity for FcγRIIB as determined based on an ELISAassay and/or an ADCC based assay using ch-4-4-20 antibody carrying thevariant Fc region comprises a substitution at position 379 withmethionine; at position 219 with tyrosine; at position 282 withmethionine; at position 401 with valine; at position 222 withasparagine; at position 334 with isoleucine; at position 334 withglutamic acid; at position 275 with tyrosine; at position 398 withvaline.

The invention encompasses a molecule comprising a variant Fc region,wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculespecifically binds FcγRIIB with about a 3 fold lower affinity than acomparable molecule comprising the wild-type Fc region as determined byan ELISA assay, wherein said at least one amino acid modificationcomprises substitution at position 288 with asparagine, at position 330with serine, and at position 396 with leucine. The invention encompassesa molecule comprising a variant Fc region, wherein said variant Fcregion comprises at least one amino acid modification relative to awild-type Fc region, such that said molecule specifically binds FcγRIIBwith about a 10-15 fold lower affinity than a comparable moleculecomprising the wild-type Fc region as determined by an ELISA assay,wherein said at least one amino acid modification comprises substitutionat position 316 with aspartic acid, at position 378 with valine, and atposition 399 with glutamic acid. The invention encompasses a moleculecomprising a variant Fc region, wherein said variant Fc region comprisesat least one amino acid modification relative to a wild-type Fc region,such that said molecule specifically binds FcγRIIB with about a 10 foldlower affinity than a comparable molecule comprising the wild-type Fcregion as determined by an ELISA assay, wherein said at least one aminoacid modification comprises substitution at position 315 withisoleucine, at position 379 with methionine, and at position 399 withglutamic acid. The invention encompasses a molecule comprising a variantFc region, wherein said variant Fc region comprises at least one aminoacid modification relative to a wild-type Fc region, such that saidmolecule specifically binds FcγRIIB with about a 7 fold lower affinitythan a comparable molecule comprising the wild-type Fc region asdetermined by an ELISA assay, wherein said at least one amino acidmodification comprises substitution at position 243 with isoleucine, atposition 379 with leucine, and at position 420 with valine. Theinvention encompasses a molecule comprising a variant Fc region, whereinsaid variant Fc region comprises at least one amino acid modificationrelative to a wild-type Fc region, such that said molecule specificallybinds FcγRIIB with about a 3 fold lower affinity than a comparablemolecule comprising the wild-type Fc region as determined by an ELISAassay, wherein said at least one amino acid modification comprisessubstitution at position 392 with threonine and at position 396 withleucine. The invention encompasses a molecule comprising a variant Fcregion, wherein said variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that said moleculespecifically binds FcγRIIB with about a 5 fold lower affinity than acomparable molecule comprising the wild-type Fc region as determined byan ELISA assay, wherein said at least one amino acid modificationcomprises substitution at position 268 with asparagine and at position396 with leucine. The invention also encompasses a molecule comprising avariant Fc region, wherein said variant Fc region comprises at least oneamino acid modification relative to a wild-type Fc region, such thatsaid molecule specifically binds FcγRIIB with about a 2 fold loweraffinity than a comparable molecule comprising the wild-type Fc regionas determined by an ELISA assay, wherein said at least one amino acidmodification comprises substitution at position 319 with phenylalanine,at position 352 with leucine, and at position 396 with leucine.

C. Mutants with Enhanced Affinity to FcγRIIIA and FcγRIIB

The invention encompasses molecules comprising variant Fc regions,having one or more amino acid modifications, which modificationsincrease the affinity of the variant Fc region for FcγRIIIA and FcγRIIBby at least 65%, at least 70%, at least 75%, at least 85%, at least 90%,at least 95%, at least 99%, at least 100%, at least 200%, at least 300%,at least 400% and decreases the affinity of the variant Fc region forFcγRIIB by at least 65%, at least 70%, at least 75%, at least 85%, atleast 90%, at least 95%, at least 99%, at least 100%, at least 200%, atleast 300%, at least 400%. In a specific embodiment, the molecule of theinvention comprising a variant Fc region with an enhanced affinity forFcγRIIIA and an enhanced affinity for FcγRIIB (as determined based on anELISA assay and/or an ADCC based assay using ch-4-4-20 antibody carryingthe variant Fc region as described herein) comprises a substitution atposition 415 with isoleucine and at position 251 with phenylalanine; ora substitution at position 399 with glutamic acid, at position 292 withleucine, and at position 185 with methionine; or a substitution atposition 408 with isoleucine, at position 215 with isoleucine, and atposition 125 with leucine; or a substitution at position 385 withglutamic acid and at position 247 with histidine; or a substitution atposition 348 with methionine, at position 334 with asparagine, atposition 275 with isoleucine, at position 202 with methionine and atposition 147 with threonine; or a substitution at position 246 withthreonine and at position 396 with histidine; or a substitution atposition 268 with aspartic acid and at position 318 with aspartic acid;or a substitution at position 288 with asparagine, at position 330 withserine and at position 396 with leucine; or a substitution at position244 with histidine, at position 358 with methionine, at position 379with methionine, at position 384 with lysine and at position 397 withmethionine; or a substitution at position 217 with serine, at position378 with valine, and at position 408 with arginine; or a substitution atposition 247 with leucine, at position 253 with asparagine, and atposition 334 with asparagine; or a substitution at position 246 withisoleucine and at position 334 with asparagine; or a substitution atposition 320 with glutamic acid and at position 326 with glutamic acid;or a substitution at position 375 with cysteine and at position 396 withleucine; or a substitution at position 343 with serine, at position 353with leucine, at position 375 with isoleucine, at position 383 withasparagine; or a substitution at position 394 with methionine and atposition 397 with methionine; or a substitution at position 216 withaspartic acid, at position 345 with lysine and at position 375 withisoleucine; or a substitution at position 288 with asparagine, atposition 330 with serine, and at position 396 with leucine; or asubstitution at position 247 with leucine and at position 389 withglycine; or a substitution at position 222 with asparagine, at position335 with asparagine, at position 370 with glutamic acid, at position 378with valine and at position 394 with methionine; or a substitution atposition 316 with aspartic acid, at position 378 with valine and atposition 399 with glutamic acid; or a substitution at position 315 withisoleucine, at position 379 with methionine, and at position 394 withmethionine; or a substitution at position 290 with threonine and atposition 371 with aspartic acid; or a substitution at position 247 withleucine and at position 398 with glutamine; or a substitution atposition 326 with glutamine; at position 334 with glutamic acid, atposition 359 with asparagine, and at position 366 with serine; or asubstitution at position 247 with leucine and at position 377 withphenylalanine; or a substitution at position 378 with valine, atposition 390 with isoleucine and at position 422 with isoleucine; or asubstitution at position 326 with glutamic acid and at position 385 withglutamic acid; or a substitution at position 282 with glutamic acid, atposition 369 with isoleucine and at position 406 with phenylalanine; ora substitution at position 397 with methionine; at position 411 withalanine and at position 415 with asparagine; or a substitution atposition 223 with isoleucine, at position 256 with serine and atposition 406 with phenylalanine; or a substitution at position 298 withasparagine and at position 407 with arginine; or a substitution atposition 246 with arginine, at position 298 with asparagine, and atposition 377 with phenylalanine; or a substitution at position 235 withproline, at position 382 with methionine, at position 304 with glycine,at position 305 with isoleucine, and at position 323 with isoleucine; ora substitution at position 247 with leucine, at position 313 witharginine, and at position 388 with glycine; or a substitution atposition 221 with tyrosine, at position 252 with isoleucine, at position330 with glycine, at position 339 with threonine, at position 359 withasparagine, at position 422 with isoleucine, and at position 433 withleucine; or a substitution at position 258 with aspartic acid, and atposition 384 with lysine; or a substitution at position 241 with leucineand at position 258 with glycine; or a substitution at position 370 withasparagine and at position 440 with asparagine; or a substitution atposition 317 with asparagine and a deletion at position 423; or asubstitution at position 243 with isoleucine, at position 379 withleucine and at position 420 with valine; or a substitution at position227 with serine and at position 290 with glutamic acid; or asubstitution at position 231 with valine, at position 386 withhistidine, and at position 412 with methionine; or a substitution atposition 215 with proline, at position 274 with asparagine, at position287 with glycine, at position 334 with asparagine, at position 365 withvaline and at position 396 with leucine; or a substitution at position293 with valine, at position 295 with glutamic acid and at position 327with threonine; or a substitution at position 319 with phenylalanine, atposition 352 with leucine, and at position 396 with leucine; or asubstitution at position 392 with threonine and at position 396 withleucine; at a substitution at position 268 with asparagine and atposition 396 with leucine; or a substitution at position 290 withthreonine, at position 390 with isoleucine, and at position 396 withleucine; or a substitution at position 326 with isoleucine and atposition 396 with leucine; or a substitution at position 268 withaspartic acid and at position 396 with leucine; or a substitution atposition 210 with methionine and at position 396 with leucine; or asubstitution at position 358 with proline and at position 396 withleucine; or a substitution at position 288 with arginine, at position307 with alanine, at position 344 with glutamic acid, and at position396 with leucine; or a substitution at position 273 with isoleucine, atposition 326 with glutamic acid, at position 328 with isoleucine and atposition 396 with leucine; or a substitution at position 326 withisoleucine, at position 408 with asparagine and at position 396 withleucine; or a substitution at position 334 with asparagine and atposition 396 with leucine; or a substitution at position 379 withmethionine and at position 396 with leucine; or a substitution atposition 227 with serine and at position 396 with leucine; or asubstitution at position 217 with serine and at position 396 withleucine; or a substitution at position 261 with asparagine, at position210 with methionine and at position 396 with leucine; or a substitutionat position 419 with histidine and at position 396 with leucine; or asubstitution at position 370 with glutamic acid and at position 396 withleucine; or a substitution at position 242 with phenylalanine and atposition 396 with leucine; or a substitution at position 255 withleucine and at position 396 with leucine; or a substitution at position240 with alanine and at position 396 with leucine; or a substitution atposition 250 with serine and at position 396 with leucine; or asubstitution at position 247 with serine and at position 396 withleucine; or a substitution at position 410 with histidine and atposition 396 with leucine; or a substitution at position 419 withleucine and at position 396 with leucine; or a substitution at position427 with alanine and at position 396 with leucine; or a substitution atposition 258 with aspartic acid and at position 396 with leucine; or asubstitution at position 384 with lysine and at position 396 withleucine; or a substitution at position 323 with isoleucine and atposition 396 with leucine; or a substitution at position 244 withhistidine and at position 396 with leucine; or a substitution atposition 305 with leucine and at position 396 with leucine; or asubstitution at position 400 with phenylalanine and at position 396 withleucine; or a substitution at position 303 with isoleucine and atposition 396 with leucine; or a substitution at position 243 withleucine, at position 305 with isoleucine, at position 378 with asparticacid, at position 404 with serine and at position 396 with leucine; or asubstitution at position 290 with glutamic acid, at position 369 withalanine, at position 393 with alanine and at position 396 with leucine;or a substitution at position 210 with asparagine, at position 222 withisoleucine, at position 320 with methionine and at position 396 withleucine; or a substitution at position 217 with serine, at position 305with isoleucine, at position 309 with leucine, at position 390 withhistidine and at position 396 with leucine; or a substitution atposition 246 with asparagine; at position 419 with arginine and atposition 396 with leucine; or a substitution at position 217 withalanine, at position 359 with alanine and at position 396 with leucine;or a substitution at position 215 with isoleucine, at position 290 withvaline and at position 396 with leucine; or a substitution at position275 with leucine, at position 362 with histidine, at position 384 withlysine and at position 396 with leucine; or a substitution at position334 with asparagine; or a substitution at position 400 with proline; ora substitution at position 407 with isoleucine; or a substitution atposition 372 with tyrosine; or a substitution at position 366 withasparagine; or a substitution at position 414 with asparagine; or asubstitution at position 352 with leucine; or a substitution at position225 with serine; or a substitution at position 377 with asparagine; or asubstitution at position 248 with methionine.

E. Mutants with Altered FcγR-Mediated Effector Functions

The invention encompasses molecules, e.g., immunoglobulins comprising Fcvariants with altered effector functions, preferably, added effectorfunctions, i.e., where the variants exhibit detectable levels of one ormore effector functions that are not detectable in the parent antibody.In some embodiments, immunoglobulins comprising Fc variants mediateeffector function more effectively in the presence of effector cells asdetermined using assays known in the art and exemplified herein. Inspecific embodiments, the Fc variants of the invention may be combinedwith other known Fc modifications that enhance effector function, suchthat the combination has an additive, synergistic effect. The Fcvariants of the invention have conferred or enhanced effector functionin vitro and/or in vivo.

In a specific embodiment, the immunoglobulins of the invention have anenhanced FcγR-mediated effector function as determined using ADCCactivity assays disclosed herein. Examples of effector functions thatcould be mediated by the molecules of the invention include, but are notlimited to, C1q binding, complement-dependent cytotoxicity,antibody-dependent cell mediate cytotoxicity (ADCC), phagocytosis, etc.The effector functions of the molecules of the invention can be assayedusing standard methods known in the art, examples of which are disclosedin Section 5.2.7. In a specific embodiment, the immunoglobulins of theinvention comprising a variant Fc region mediate ADCC where the parentmolecule does not exhibit detectable levels of ADCC activity or inducesADCC 2-fold more effectively, than an immunoglobulin comprising awild-type Fc region. In other embodiments, the immunoglobulins of theinvention comprising a variant Fc region mediate ADCC where the parentmolecule does not exhibit detectable levels of ADCC activity or inducesADCC at least 4-fold, at least 8-fold, at least 10-fold, at least100-fold, at least 1000-fold, at least 10⁴-fold, at least 10⁵-fold moreeffectively, than an immunoglobulin comprising a wild-type Fc region. Inanother specific embodiment, the immunoglobulins of the invention havealtered C1q binding activity. In some embodiments, the immunoglobulinsof the invention mediate C1q binding activity where the parent moleculedoes not exhibit detectable levels of C1q binding activity or has atleast 2-fold, at least 4-fold, at least 8-fold, at least 10-fold, atleast 100-fold, at least 1000-fold, at least 10⁴-fold, at least 10⁵-foldhigher C1q binding activity than an immunoglobulin comprising awild-type Fc region. In yet another specific embodiment, theimmunoglobulins of the invention have altered complement dependentcytotoxicity. In yet another specific embodiment, the immunoglobulins ofthe invention have complement dependent cytotoxicity where the parentmolecule does not exhibit detectable levels of complement dependentcytotoxicity or enhances complement dependent cytotoxicity to levelsgreater than an immunoglobulin comprising a wild-type Fc region. In someembodiments, the immunoglobulins of the invention have at least 2-fold,at least 4-fold, at least 8-fold, at least 10-fold, at least 100-fold,at least 1000-fold, at least 10⁴-fold, at least 10⁵-fold highercomplement dependent cytotoxicity than an immunoglobulin comprising awild-type Fc region.

In other embodiments, immunoglobulins of the invention have phagocytosisactivity where the parent molecule does not exhibit detectable levels ofphagocytosis activity or have enhanced phagocytosis activity relative toan immunoglobulin comprising a wild-type Fc region, as determined bystandard assays known to one skilled in the art or disclosed herein. Insome embodiments, the immunoglobulins of the invention have at least2-fold, at least 4-fold, at least 8-fold, at least 10-fold higherphagocytosis activity relative to an immunoglobulin comprising awild-type Fc region.

In a specific embodiment, the invention encompasses an immunoglobulincomprising a variant Fc region with one or more amino acidmodifications, such that the immunoglobulin has an effector function,e.g., antibody dependent cell mediated cytotoxicity or phagocytosis,where the parent molecule does not exhibit detectable levels of theeffector function or has an enhanced effector function. In a specificembodiment, the one or more amino acid modifications which increase theADCC activity of the immunoglobulin comprise a substitution at position379 with methionine; or a substitution at position 243 with isoleucineand at position 379 with leucine; or a substitution at position 288 withasparagine, at position 330 with serine, and at position 396 withleucine; or a substitution at position 243 leucine and at position 255with leucine; or a substitution at position 334 with glutamic acid, atposition 359 with asparagine, and at position 366 with serine; or asubstitution at position 288 with methionine and at position 334 withglutamic acid; or a substitution at position 334 with glutamic acid andat position 292 with leucine; or a substitution at position 316 withaspartic acid, at position 378 with valine, and at position 399 withglutamic acid; or a substitution at position 315 with isoleucine, atposition 379 with methionine, and at position 399 with glutamic acid; ora substitution at position 243 with isoleucine, at position 379 withleucine, and at position 420 with valine; or a substitution at position247 with leucine and at position 421 with lysine; or a substitution atposition 248 with methionine; or a substitution at position 392 withthreonine and at position 396 with leucine; or a substitution atposition 293 with valine, at position 295 with glutamic acid, and atposition 327 with threonine; or a substitution at position 268 withasparagine and at position 396 with leucine; or a substitution atposition 319 with phenylalanine, at position 352 with leucine, and atposition 396 with leucine; or a substitution at position 255 withleucine, at position 396 with leucine, at position 270 with glutamicacid, and at position 300 with leucine; or a substitution at position240 with alanine, at position 396 with leucine, and at position 270 withglutamic acid; or a substitution at position 370 with glutamic acid, atposition 396 with leucine, and at position 270 with glutamic acid; or asubstitution at position 392 with threonine, at position 396 withleucine, and at position 270 with glutamic acid; or a substitution atposition 370 with glutamic acid and at position 396 with leucine; or asubstitution at position 419 with histidine and at position 396 withleucine; or a substitution at position 255 with leucine, at position 396with leucine, at position 270 with glutamic acid, and at position 292with glycine. In other specific embodiments, the variant Fc region has aleucine at position 247, a lysine at position 421 and a glutamic acid atposition 270 (MgFc31/60); a threonine at position 392, a leucine atposition 396, a glutamic acid at position 270, and a leucine at position243 (MgFc38/60/F243L); a histidine at position 419, a leucine atposition 396, and a glutamic acid at position 270 (MGFc51/60); ahistidine at position 419, a leucine at position 396, a glutamic acid atposition 270, and a leucine at position 243 (MGFc51/60/F243L); analanine at position 240, a leucine at position 396, and a glutamic acidat position 270 (MGFc52/60); a lysine at position 255 and a leucine atposition 396 (MgFc55); a lysine at position 255, a leucine at position396, and a glutamic acid at position 270 (MGFc55/60); a lysine atposition 255, a leucine at position 396, a glutamic acid at position270, and a lysine at position 300 (MGFc55/60/Y300L); a lysine atposition 255, a leucine at position 396, a glutamic acid at position270, and a glycine at position 292 (MGFc55/60/R292G); a lysine atposition 255, a leucine at position 396, a glutamic acid at position270, and a leucine at position 243 (MgFc55/60/F243L); a glutamic acid atposition 370, a leucine at position 396, and a glutamic acid at position270 (MGFc59/60); a glutamic acid at position 270, an aspartic acid atposition 316, and a glycine at position 416 (MgFc71); a leucine atposition 243, a proline at position 292, an isoleucine at position 305,and a leucine at position 396 (MGFc74/P396L); or a leucine at position243, a glutamic acid at position 270, an asparagine at position 392 anda leucine at position 396; or a leucine at position 243, a leucine atposition 255, a glutamic acid at position 270 and a leucine at position396; or a glutamine at position 297.

In another specific embodiment, the one or more amino acid modificationswhich confers or increases the ADCC activity of the immunoglobulin isany of the mutations listed below, in table 7.

TABLE 7 AMINO ACID MODIFICATIONS WHICH CONFER OR INCREASE ADCC E333A,K334A R292L, K334E V379M S219Y V282M K222N F243I, V379L F243L, R255L,E318K K334I K334E, T359N, T366S K288M, K334E K288N, A330S, P396L K326EG316D, A378V, D399E N315I, V379M, T394M F243I, V379L, G420V E293V,Q295E, A327T Y319F, P352L, P396L K392T, P396L K248M H268N, P396L K290T,N390I, P396L K326I, P396L H268D, P396L K210M, P396L L358P, P396L K288R,T307A, K344E, P396L V273I, K326E, L328I, P396L K326I, S408N, P396LK334N, P396L V379M, P396L P227S, P396L P217S, P396L K261N, K210M, P396LQ419H, P396L K370E, P396L L242F, P396L F243L, V305I, A378D, F404S, P396LR255L, P396L V240A, P396L T250S, P396L P247S, P396L K290E, V369A, T393A,P396L K210N, K222I, K320M, P396L L410H, P396L Q419L, P396L V427A, P396LP217S, V305I, I309L, N390H, P396L E258D, P396L N384K, P396L V323I, P396LK246N, Q419R, P396L P217A, T359A, P396L P244H, P396L V215I, K290V, P396LF275L, Q362H, N384K, P396L V305L, P396L S400F, P396L V303I, P396L D270E,G316D, R416G P247L, N421K P247L, N421K, D270E Q419H, P396L, D270E K370E,P396L, D270E R255L, P396L, D270E V240A, P396L, D270E K392T, P396L, D270ER255L, P396L, D270E, Y300L R255L, P396L, D270E, R292G K392T, P396L,D270E, F243L Q419H, P396L, D270E, F243L R255L, P396L, D270E, F243LF243L, D270E, K392N, P396L F243L, R255L, D270E, P396L

Alternatively or additionally, it may be useful to combine the aboveamino acid modifications or any other amino acid modifications disclosedherein with one or more further amino acid modifications that conferand/or alter C1q binding and/or complement dependent cytoxicity functionof the Fc region. The further amino acid substitutions described hereinwill generally serve to alter the ability of the starting molecule tobind to C1q and/or modify its complement dependent cytotoxicityfunction, e.g., to reduce and preferably abolish these effectorfunctions. Molecules comprising substitutions at one or more of thedescribed positions with conferred or improved C1q binding and/orcomplement dependent cytotoxicity (CDC) function are contemplatedherein. For example, the starting molecule may be unable to bind C1qand/or mediate CDC and may be modified according to the teachings hereinsuch that it acquires these further effector functions. Moreover,molecules with preexisting C1q binding activity, optionally furtherhaving the ability to mediate CDC may be modified such that one or bothof these activities are enhanced.

As disclosed above, one can design an Fc region with altered effectorfunction, e.g., by modifying or conferring C1q binding and/or FcRbinding and thereby changing CDC activity and/or ADCC activity. Forexample, one can generate a variant Fc region with improved or conferredC1q binding and improved or conferred FcγRIII binding; e.g., having bothconferred or improved ADCC activity and conferred or improved CDCactivity. Alternatively, where one desires that effector function bereduced or ablated, one may engineer a variant Fc region with reducedCDC activity and/or reduced ADCC activity. In other embodiments, one mayincrease only one of these activities, and optionally also reduce theother activity, e.g., to generate an Fc region variant with improvedADCC activity, but reduced CDC activity and vice versa.

The invention encompasses molecules with specific variants of the Fcregion that have been identified using the methods of the invention froma yeast library of mutants after 2nd-4th-round of sorting are listed inTable 8. Table 8 summarizes the various mutants that were identifiedusing the methods of the invention. The mutants were assayed using anELISA assay for determining binding to FcγRIIIA and FcγRIIB. The mutantswere also tested in an ADCC assay, by cloning the Fc variants into a ch4-4-20 antibody using methods disclosed and exemplified herein. Boldeditems refer to experiments, in which the ch4-4-20 were purified priorthe ADCC assay. The antibody concentration used was standard for ADCCassays, in the range 0.5 μg/mL-1.0 μg/mL.

TABLE 8 MUTATIONS IDENTIFIED IN THE Fc REGION Binding Binding to to4-4-20 ADCC FcγRIIIA FcγRIIB (Relative Mutations Domain (ELISA) (ELISA)Lysis (Mut/Wt) pYD-CH1 library FACS screen with 3A tetramer Q347H; A339VCH3 ↑□0.5x NT S415I; L251F CH2, CH3 ↑□0.5x ↑.75x 0.82 K392R CH3 N/C NTD399E; R292L; V185M CH1, CH2, CH3 N/C ↑□0.5x 0.65 0.9 K290E; L142P CH1,CH2 N/C NT R301C; M252L; S192T CH1, CH2 ↓.5x NT P291S; K288E; H268L;A141V CH1, CH2 ↓.5x NT N315I CH2 N/C ↑.75x S132I CH1 N/C NT S383N;N384K; T256N; V262L; K218E; R214I; K205E; All ↑0.5x NT F149Y; K133MS408I; V215I; V125L CH1, CH2, CH3 ↑□0.5x ↑.75x 0.62 P396L CH3 ↑1x ↑1x0.55 G385E; P247H; CH2, CH3 ↑1x ↑.75x 0.44 P396H CH3 ↑1x □↑1x 0.58 A162VCH1 N/C NT V348M; K334N; F275I; Y202M; K147T CH1, CH2, CH3 ↑□0.5x ↑.75x0.33 H310Y; T289A; G337E CH2 ↑.5x NT S119F; G371S; Y407V; E258D CH1,CH2, CH3 N/C N/C 0.29 K409R; S166N CH1, CH3 N/C NT in vitro SiteDirected mutants R292L CH2 NT NT 0.82 T359N CH3 NT NT 1.06 T366S CH3 NTNT 0.93 E333A, K334A CH2 NT NT 1.41 R292L, K334E CH2 NT NT 1.41; 1.64R292L, P396L, T359N CH2, CH3 NT NT 0.89; 1.15 V379L CH3 NT NT 0.83 K288NCH2 NT NT 0.78 A330S CH2 NT NT 0.52 F243L CH2 NT NT 0.38 E318K CH2 NT NT0.86 K288N, A330S CH2 NT NT 0.08 R255L, E318K CH2 NT NT 0.82 F243L,E318K CH2 NT NT 0.07 Mutants in 4-4-20 mini-library Increased FcγRIIIAbinding, decreased or no change to FcγRIIB binding N/C means no change;N/B means no binding; NT means not tested V379M CH3 ↑2x N/C 1.47 S219YHinge ↑1x ↓ or N/B 1.28 V282M CH2 ↑1x ↓ or N/B 1.25; 1 F275I, K334N,V348M CH2 ↑0.5x N/C D401V CH3 ↑ 0.5x N/C V279L, P395S CH2 ↑1x N/C K222NHinge ↑1x ↓or N/B 1.33; 0.63 K246T, Y319F CH2 ↑1x N/C F243I, V379L CH2,CH3 ↑1.5x ↓ or N/B 1.86; 1.35 F243L, R255L, E318K CH2 ↑1x ↓ or N/B 1.81;1.45 K334I CH2 ↑1x N/C 2.1; 1.97 K334E, T359N, T366S CH2, CH3 ↑1.5x N/C1.49; 1.45 K288M, K334E CH2 ↑ 3x ↓ or N/B 1.61; 1.69 K334E, E380D CH2,CH3 ↑1.5x N/C T256S, V305I, K334E, N390S CH2, CH3 ↑1.5x N/C K334E CH2↑2.5x N/C 1.75; 2.18 T335N, K370E, A378V, T394M, S424L CH2, CH3 ↑0.5xN/C E233D, K334E CH2 ↑1.5x N/C 0.94; 1.02 K334E, T359N, T366S, Q386R CH2↑1x N/C Increased Binding to FcγIIIA and FcγRIIB K246T, P396H CH2, CH3↑1x ↑ 2.5x H268D, E318D CH2 ↑1.5x ↑ 5x K288N, A330S, P396L CH2, CH3 ↑ 5x↑ 3x 2.34; 1.66; 2.54 I377F CH3 ↑1.5x ↑0.5x P244H, L358M, V379M, N384K,V397M CH2, CH3 ↑1.75x ↑1.5x P217S, A378V, S408R Hinge, CH3 ↑ 2x ↑4.5xP247L, I253N, K334N CH2 ↑ 3x ↑ 2.5x P247L CH2 ↑0.5x ↑ 4x 0.91; 0.84F372Y CH3 ↑0.75x ↑5.5x 0.88; 0.59 K326E CH2 ↑ 2x ↑ 3.5x 1.63; 2 K246I,K334N CH2 ↑0.5x ↑ 4x 0.66; 0.6 K320E, K326E CH2 ↑1x ↑1x H224L Hinge↑0.5x ↑ 5x 0.55; 0.53 S375C, P396L CH3 ↑1.5x ↑4.5x D312E, K327N, I378SCH2, CH3 ↑0.5x N/C K288N, K326N CH2 ↑1x N/C F275Y CH2 ↑ 3x N/C 0.64P247L, N421K CH2, CH3 ↑ 3x N/C 2.0 S298N, W381R CH2, CH3 ↑ 2x N/C D280E,S354F, A431D, L441I CH2, CH3 ↑ 3x N/C 0.62 R255Q, K326E CH2 ↑ 2x N/C0.79 K218R, G281D, G385R H, CH2, CH3 ↑3.5x N/C 0.67 L398V CH3 ↑1.5x N/CP247L, A330T, S440G CH2, CH3 ↑0.75x ↓ 0.25x V284A, F372L CH2, CH3 1x N/CT335N, P387S, H435Q CH2, CH3 1.25x N/C P247L, A431V, S442F CH2, CH3 1xN/C Increased Binding to FcγRIIIA and FcγRIIB P343S, P353L, S375I, S383NCH3 ↑ 0.5x ↑ 6x T394M, V397M CH3 ↑0.5x ↑ 3x E216D, E345K, S375I H, CH2,CH3 ↑ 0.5x ↑ 4x K334N. CH2 ↑0.5x ↑ 2x K288N, A330S, P396L CH2, CH3 ↑0.5x↑ 9x P247L, E389G CH2, CH3 ↑1.5x ↑ 9x K222N, T335N, K370E, A378V, T394MH, CH2, CH3 ↑1x ↑ 7x G316D, A378V, D399E CH2, CH3 ↑1.5x ↑14x 2.24 N315I,V379M, T394M CH2, CH3 ↑1x ↑ 9x 1.37 K290T, G371D, CH2, CH3 ↑ 0.25x ↑ 6xP247L, L398Q CH2, CH3 ↑1.25x ↑10x K326Q, K334E, T359N, T366S CH2, CH3↑1.5x ↑ 5x S400P CH3 ↑1x ↑ 6x P247L, I377F CH2, CH3 ↑1x ↑ 5x A378V,N390I, V422I CH3 ↑ 0.5x ↑ 5x K326E, G385E CH2, CH3 ↑0.5x ↑15x V282E,V369I, L406F CH2, CH3 ↑ 0.5x ↑ 7x V397M, T411A, S415N CH3 ↑ 0.25x ↑5xT223I, T256S, L406F H, CH2, CH3 ↑ 0.25x ↑ 6x S298N, S407R CH2, CH3 ↑0.5x↑ 7x K246R, S298N, I377F CH2, CH3 ↑1x ↑ 5x S407I CH3 ↑ 0.5x ↑4x F372YCH3 ↑0.5x ↑4x L235P, V382M, S304G, V305I, V323I CH2, CH3 ↑ 2x ↑ 2xP247L, W313R, E388G CH2, CH3 ↑1.5x ↑1x D221Y, M252I, A330G, A339T,T359N, V422I, H433L H, CH2, CH3 ↑2.5x ↑ 6x E258D, N384K CH2, CH3 ↑1.25x↑4x F241L, E258G CH2 ↑ 2x ↑ 2.5x −0.08 K370N, S440N CH3 ↑1x ↑ 3.5xK317N, F423-deleted CH2, CH3 ↑ 2.5x ↑ 7x 0.18 F243I, V379L, G420V CH2,CH3 ↑ 2.5x ↑3.5x 1.35 P227S, K290E H, CH2 ↑1x ↑ 0.5x A231V, Q386H, V412MCH2, CH3 ↑1.5x ↑ 6x T215P, K274N, A287G, K334N, L365V, P396L H, CH2, CH3↑2x ↑ 4x Increased Binding to FcγRIIB but not FcγRIIIA K334E, E380D CH2,CH3 N/C ↑4.5x T366N CH3 N/C ↑ 5x P244A, K326I, C367R, S375I, K447T CH2,CH3 N/C ↑ 3x C229Y, A287T, V379M, P396L, L443V H, CH2, CH3 ↓ 0.25x ↑10xDecreased binding to FcγRIIIA and FcγRIIB R301H, K340E, D399E CH2, CH3 ↓0.50x ↓ 0.25x K414N CH3 ↓ 0.25x N/B P291S, P353Q CH2, CH3 ↓ 0.50x ↓0.25x V240I, V281M CH2 ↓ 0.25x ↓ 0.25x P232S, S304G CH2 N/B N/B E269K,K290N, Q311R, H433Y CH2, CH3 N/B N/B M352L CH3 N/B N/B E216D, K334R,S375I H, CH2, CH3 N/B N/B P247L, L406F CH2, CH3 N/B N/B T335N, P387S,H435Q CH2, CH3 N/B N/B T225S CH2 ↓ 0.25x ↓ 0.50x D399E, M428L CH3 ↓0.50x ↓ 0.50x K246I, Q362H, K370E CH2, CH3 N/B ↓ 0.50x K334E, E380D,G446V CH2, CH3 N/B N/B I377N CH3 ↓ 0.50x N/B V303I, V369F, M428L CH2,CH3 N/B N/B L251F, F372L CH2, CH3 N/B N/B K246E, V284M, V308A CH2, CH3N/B N/B D399E, G402D CH3 N/B N/B D399E, M428L CH3 N/B N/B FcγRIIBdepletion/FcγRIIIA selection: Naive Fc library. E293V, Q295E, A327T CH2↑0.4x ↓ or N/B 4.29 Y319F, P352L, P396L CH2, CH3 ↑3.4x ↑2x 1.09 K392T,P396L CH3 ↑ 4.5x ↑ 2.5x 3.07 K248M CH2 ↑0.4x ↓ or N/B 4.03 H268N, P396LCH2, CH3 ↑ 2.2x ↑ 4.5x 2.24 Solution competition 40X FcγRIIB-G2: P396LLibrary D221E, D270E, V308A, Q311H, P396L, G402D ↑3.6x ↑0.1x 3.17Equilibrium Screen: 0.8 μM FcγRIIIA monomer: P396L library K290T, N390I,P396L CH2, CH3 □↑2.8x ↑ 6.1x 1.93 K326I, P396L CH2, CH3 □↑2.9x ↑ 5.9x1.16 H268D, P396L CH2, CH3 ↑3.8x ↑13.7x 2.15 K210M, P396L CH1, CH3 ↑1.9x↑ 4.6x 2.02 L358P, P396L CH3 ↑1.9x ↑ 4.2x 1.58 K288R, T307A, K344E,P396L CH2, CH3 ↑ 4.1x ↑ 2.3x 3.3 V273I, K326E, L328I, P396L CH2, CH3↑1.3x ↑10.8x 0.78 K326I, S408N, P396L CH2, CH3 ↑4x ↑ 9.3x 1.65 K334N,P396L CH2, CH3 ↑3.1x ↑ 3x 2.43 V379M, P396L CH3 ↑1.9x ↑5.6x 2.01 P227S,P396L CH2, CH3 ↑1.5x ↑ 4x 2.01 P217S, P396L H, CH3 ↑1.6x ↑4.5x 2.04K261N, K210M, P396L CH2, CH3 ↑ 2x ↑ 4.2x 2.06 Kinetic Screen: O.8 μM, 1′with cold 8 μM FcγRIIIA: P396L Library term is M, P396L CH3 ↑1.9x ↑ 7.2x3.09 Q419H, P396L CH3 ↑ 2x ↑ 6.9x 2.24 K370E, P396L CH3 ↑2x ↑6.6x 2.47L242F, P396L CH2, CH3 ↑ 2.5x ↑ 4.1x 2.4 F243L, V305I, A378D, F404S,P396L CH2, CH3 ↑1.6x ↑5.4x 3.59 R255L, P396L CH2, CH3 ↑1.8x ↑ 6x 2.79V240A, P396L CH2, CH3 ↑1.3x ↑ 4.2x 2.35 T250S, P396L CH2, CH3 ↑1.5x↑6.8x 1.60 P247S, P396L CH2, CH3 ↑1.2x ↑ 4.2x 2.10 K290E, V369A, T393A,P396L CH2, CH3 ↑1.3x ↑ 6.7x 1.55 K210N, K222I, K320M, P396L H, CH2, CH3↑ 2.7x ↑ 8.7x 1.88 L410H, P396L CH3 ↑1.7x ↑ 4.5x 2.00 Q419L, P396L CH3 ↑2.2x ↑ 6.1x 1.70 V427A, P396L CH3 ↑1.9x ↑4.7x 1.67 P217S, V305I, I309L,N390H, P396L H, CH2, CH3 ↑2x ↑ 7x 1.54 E258D, P396L CH2, CH3 ↑1.9x ↑4.9x 1.54 N384K, P396L CH3 ↑ 2.2x ↑5.2x 1.49 V323I, P396L CH2, CH3 ↑1.1x↑ 8.2x 1.29 K246N, Q419R, P396L CH2, CH3 ↑1.1x ↑ 4.8x 1.10 P217A, T359A,P396L H, CH2, CH3 ↑1.5x ↑ 4.8x 1.17 P244H, P396L CH2, CH3 ↑2.5x ↑ 4x1.40 V215I, K290V, P396L H, CH2, CH3 ↑2.2x ↑ 4.6x 1.74 F275L, Q362H,N384K, P396L CH2, CH3 ↑ 2.2x ↑ 3.7x 1.51 V305L, P396L CH2, CH3 ↑1.3x ↑5.5x 1.50 S400F, P396L CH3 ↑1.5x ↑4.7x 1.19 V303I, P396L CH3 ↑1.1x ↑ 4x1.01 FcγRIIB depletion FcγRIIIA 158V solid phase selection: NaïveLibrary A330V, H433Q, V427M CH2, CH3 NT NT NT V263Q, E272D, Q419H CH2,CH3 NT NT NT N276Y, T393N, W417R CH2, CH3 NT NT NT V282L, A330V, H433Y,T436R CH2, CH3 NT NT NT A330V, Q419H CH2, CH3 NT NT NT V284M, S298N,K334E, R355W CH2, CH3 NT NT NT A330V, G427M, K438R CH2, CH3 NT NT NTS219T, T225K, D270E, K360R CH2, CH3 NT NT NT K222E, V263Q, S298N CH2 NTNT NT V263Q, E272D CH2 NT NT NT R292G CH2 NT NT NT S298N CH2 NT NT NTE233G, P247S, L306P CH2 NT NT NT D270E CH2 NT NT NT S219T, T225K, D270ECH2 NT NT NT K326E, A330T CH2 NT NT NT E233G CH2 NT NT NT S254T, A330V,N361D, P243L CH2, CH3 NT NT NT FcγRIIB depletion FcγRIIIA 158F solidphase selection: Naïve Library 158F by FACS top 0.2% V284M, S298N,K334E, R355W R416T CH2, CH3 NT NT FcγRIIB depletion FcgRIIA 131H solidphase selection: Naïve Library R292P, V305I CH2, CH2 NT NT D270E, G316D,R416G CH2, CH3 NT NT V284M, R292L, K370N CH2, CH3 NT NT R292P, V305I,F243L CH2 NT NT

In certain embodiments, the invention provides modified molecules withvariant Fc regions, having one or more amino acid modifications, whichone or more amino acid modifications confer an effector function and/orincrease the affinity of the molecule for FcγRIIIA and/or FcγRIIA. Suchmolecules include IgG molecules that naturally contain FcγR bindingregions (e.g., FcγRIIIA and/or FcγRIIB binding region), orimmunoglobulin derivatives that have been engineered to contain an FcγRbinding region (e.g., FcγRIIIA and/or FcγRIIB binding region). Themodified molecules of the invention include any immunoglobulin moleculethat binds, preferably, immunospecifically, i.e., competes offnon-specific binding as determined by immunoassays well known in the artfor assaying specific antigen-antibody binding, an antigen and containsan FcγR binding region (e.g., a FcγRIIIA and/or FcγRIIB binding region).Such antibodies include, but are not limited to, polyclonal, monoclonal,bi-specific, multi-specific, human, humanized, chimeric antibodies,single chain antibodies, Fab fragments, F(ab′)₂ fragments,disulfide-linked Fvs, Fc fusions, and fragments containing either a VLor VH domain or even a complementary determining region (CDR) thatspecifically binds an antigen, in certain cases, engineered to containor fused to an FcγR binding region.

In some embodiments, the molecules of the invention comprise portions ofan Fc region. As used herein the term “portion of an Fc region” refersto fragments of the Fc region, preferably a portion with effectoractivity and/or FcγR binding activity (or a comparable region of amutant lacking such activity). The fragment of an Fc region may range insize from 5 amino acids to the entire Fc region minus one amino acids.The portion of an Fc region may be missing up to 10, up to 20, up to 30amino acids from the N-terminus or C-terminus.

The IgG molecules of the invention are preferably IgG1 subclass of IgGs,but may also be any other IgG subclasses of given animals. For example,in humans, the IgG class includes IgG1, IgG2, IgG3, and IgG4; and mouseIgG includes IgG1, IgG2a, IgG2b, IgG2c and IgG3.

The immunoglobulins (and other polypeptides used herein) may be from anyanimal origin including birds and mammals. Preferably, the antibodiesare human, rodent (e.g., mouse and rat), donkey, sheep, rabbit, goat,guinea pig, camel, horse, or chicken. As used herein, “human” antibodiesinclude antibodies having the amino acid sequence of a humanimmunoglobulin and include antibodies isolated from human immunoglobulinlibraries or from animals transgenic for one or more humanimmunoglobulin and that do not express endogenous immunoglobulins, asdescribed infra and, for example, in U.S. Pat. No. 5,939,598 byKucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide or may be specificfor heterologous epitopes, such as a heterologous polypeptide or solidsupport material. See, e.g., PCT publications WO 93/17715; WO 92/08802;WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol., 147:60-69, 1991;U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819;Kostelny et al., J. Immunol., 148:1547-1553, 1992.

Multispecific antibodies have binding specificities for at least twodifferent antigens. While such molecules normally will only bind twoantigens (i.e. bispecific antibodies, BsAbs), antibodies with additionalspecificities such as trispecific antibodies are encompassed by theinstant invention. Examples of BsAbs include without limitation thosewith one arm directed against a tumor cell antigen and the other armdirected against a cytotoxic molecule.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983); which is incorporated herein by reference inits entirety). Because of the random assortment of immunoglobulin heavyand light chains, these hybridomas (quadromas) produce a potentialmixture of 10 different antibody molecules, of which only one has thecorrect bispecific structure. Purification of the correct molecule,which is usually done by affinity chromatography steps, is rathercumbersome, and the product yields are low. Similar procedures aredisclosed in WO 93/08829, and in Traunecker et al., EMBO J.,10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when, the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986). According to anotherapproach described in WO96/27011, a pair of antibody molecules can beengineered to maximize the percentage of heterodimers which arerecovered from recombinant cell culture. The preferred interfacecomprises at least a part of the CH3 domain of an antibody constantdomain. In this method, one or more small amino acid side chains fromthe interface of the first antibody molecule are replaced with largerside chains (e.g. tyrosine or tryptophan). Compensatory “cavities” ofidentical or similar size to the large side chain(s) are created on theinterface of the second antibody molecule by replacing large amino acidside chains with smaller ones (e.g. alanine or threonine). This providesa mechanism for increasing the yield of the heterodimer over otherunwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. See, e.g., Tutt et al., 1991, J.Immunol. 147: 60, which is incorporated herein by reference.

The antibodies of the invention include derivatives that are otherwisemodified, i.e., by the covalent attachment of any type of molecule tothe antibody such that covalent attachment does not prevent the antibodyfrom binding antigen and/or generating an anti-idiotypic response. Forexample, but not by way of limitation, the antibody derivatives includeantibodies that have been modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. Any of numerous chemical modifications maybe carried out by known techniques, including, but not limited to,specific chemical cleavage, acetylation, formylation, metabolicsynthesis of tunicamycin, etc. Additionally, the derivative may containone or more non-classical amino acids.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use chimeric, humanized,or human antibodies. A chimeric antibody is a molecule in whichdifferent portions of the antibody are derived from different animalspecies, such as antibodies having a variable region derived from amurine monoclonal antibody and a constant region derived from a humanimmunoglobulin. Methods for producing chimeric antibodies are known inthe art. See e.g., Morrison, Science, 229:1202, 1985; Oi et al.,BioTechniques, 4:214 1986; Gillies et al., J. Immunol. Methods,125:191-202, 1989; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397,which are incorporated herein by reference in their entireties.Humanized antibodies are antibody molecules from non-human species thatbind the desired antigen having one or more complementarity determiningregions (CDRs) from the non-human species and framework regions andconstant domains from a human immunoglobulin molecule. Often, frameworkresidues in the human framework regions will be substituted with thecorresponding residue from the CDR donor antibody to alter, preferablyimprove, antigen binding. These framework substitutions are identifiedby methods well known in the art, e.g., by modeling of the interactionsof the CDR and framework residues to identify framework residuesimportant for antigen binding and sequence comparison to identifyunusual framework residues at particular positions. See, e.g., Queen etal., U.S. Pat. No. 5,585,089; Riechmann et al., Nature, 332:323, 1988,which are incorporated herein by reference in their entireties.Antibodies can be humanized using a variety of techniques known in theart including, for example, CDR-grafting (EP 239,400; PCT publication WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101 and 5,585,089), veneeringor resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology,28(4/5):489-498, 1991; Studnicka et al., Protein Engineering,7(6):805-814, 1994; Roguska et al., Proc Natl. Acad. Sci. USA,91:969-973, 1994), and chain shuffling (U.S. Pat. No. 5,565,332), all ofwhich are hereby incorporated by reference in their entireties.Humanized antibodies may be generated using any of the methods disclosedin U.S. Pat. Nos. 5,693,762 (Protein Design Labs), 5,693,761, (ProteinDesign Labs) 5,585,089 (Protein Design Labs), 6,180,370 (Protein DesignLabs), and U.S. Publication Nos. 20040049014, 200300229208, each ofwhich is incorporated herein by reference in its entirety.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO96/34096; WO 96/33735; and WO 91/10741, each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For an overview of thistechnology for producing human antibodies, see Lonberg and Huszar, Int.Rev. Immunol., 13:65-93, 1995. For a detailed discussion of thistechnology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g., PCTpublications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735;European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793;5,916,771; and 5,939,598, which are incorporated by reference herein intheir entireties. In addition, companies such as Abgenix, Inc.(Freemont, Calif.), Medarex (NJ) and Genpharm (San Jose, Calif.) can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope (Jespers et al., Bio/technology,12:899-903, 1988).

The invention encompasses engineering human or humanized therapeuticantibodies (e.g., tumor specific monoclonal antibodies) in the Fcregion, by modification (e.g., substitution, insertion, deletion) of atleast one amino acid residue, which modification confers an effectorfunction activity, e.g., enhanced ADCC activity, phagocytosis activity,etc., as determined by standard assays known to those skilled in theart. The engineered therapeutic antibodies may further have increasedaffinity of the Fc region for FcγRIIIA and/or FcγRIIA. In otherembodiments, the engineered therapeutic antibodies may exhibitoligomerization activity mediated by the variant Fc region. In anotherembodiment, the invention relates to engineering human or humanizedtherapeutic antibodies (e.g., tumor specific monoclonal antibodies) inthe Fc region, by modification of at least one amino acid residue, whichmodification increases the affinity of the Fc region for FcγRIIIA and/orFcγRIIA and further decreases the affinity of the Fc region for FcγRIIB.

In another specific embodiment, the invention encompasses engineering anmonoclonal antibody by modification (e.g., substitution, insertion,deletion) of at least one amino acid residue in the Fc region whichmodification confers an effector function as determined by standardassays known in the art and disclosed and exemplified herein. In anotherembodiment, modification of the monoclonal antibody increases theaffinity of the Fc region for FcγRIIIA and/or FcγRIIA. In anotherspecific embodiment, modification of the monoclonal antibody may alsofurther decrease the affinity of the Fc region for FcγRIIB.

In a specific embodiment, the invention encompasses a modified moleculecomprising an Fc chain with a substitution at position 255 with leucine,at position 396 with leucine, at position 270 with glutamic acid, and atposition 300 with leucine; or a substitution at position 419 withhistidine, at position 396 with leucine, and at position 270 withglutamic acid; or a substitution at position 240 with alanine, atposition 396 with leucine, and at position 270 with glutamic acid; or asubstitution at position 370 with glutamic acid, at position 396 withleucine, and at position 270 with glutamic acid; or a substitution atposition 392 with threonine, at position 396 with leucine, and atposition 270 with glutamic acid; or a substitution at position 370 withglutamic acid and at position 396 with leucine; or a substitution atposition 419 with histidine and at position 396 with leucine; or asubstitution at position 247 with leucine, at position 421 with lysine,and at position 270 with glutamic acid; or a substitution at position255 with leucine, at position 396 with leucine, at position 270 withglutamic acid, and at position 292 with glycine. In other specificembodiments, the variant Fc region has a leucine at position 247, alysine at position 421 and a glutamic acid at position 270 (MgFc31/60);a threonine at position 392, a leucine at position 396, a glutamic acidat position 270, and a leucine at position 243 (MgFc38/60/F243L); ahistidine at position 419, a leucine at position 396, and a glutamicacid at position 270 (MGFc51/60); a histidine at position 419, a leucineat position 396, a glutamic acid at position 270, and a leucine atposition 243 (MGFc51/60/F243L); an alanine at position 240, a leucine atposition 396, and a glutamic acid at position 270 (MGFc52/60); a lysineat position 255 and a leucine at position 396 (MgFc55); a lysine atposition 255, a leucine at position 396, and a glutamic acid at position270 (MGFc55/60); a lysine at position 255, a leucine at position 396, aglutamic acid at position 270, and a lysine at position 300(MGFc55/60/Y300L); a lysine at position 255, a leucine at position 396,a glutamic acid at position 270, and a glycine at position 292(MGFc55/60/R292G); a lysine at position 255, a leucine at position 396,a glutamic acid at position 270, and a leucine at position 243(MgFc55/60/F243L); a glutamic acid at position 370, a leucine atposition 396, and a glutamic acid at position 270 (MGFc59/60); aglutamic acid at position 270, an aspartic acid at position 316, and aglycine at position 416 (MgFc71); a leucine at position 243, a prolineat position 292, an isoleucine at position 305, and a leucine atposition 396 (MGFc74/P396L); or a leucine at position 243, a glutamicacid at position 270, an asparagine at position 392 and a leucine atposition 396; or a leucine at position 243, a leucine at position 255, aglutamic acid at position 270 and a leucine at position 396; or aglutamine at position 297.

5.1.1 Polypeptide and Antibody Conjugates

Molecules of the invention comprising variant Fc regions may berecombinantly fused or chemically conjugated (including both covalentlyand non-covalently conjugations) to heterologous polypeptides (i.e., anunrelated polypeptide; or portion thereof, preferably at least 10, atleast 20, at least 30, at least 40, at least 50, at least 60, at least70, at least 80, at least 90 or at least 100 amino acids of thepolypeptide) to generate fusion proteins. The fusion does notnecessarily need to be direct, but may occur through linker sequences.

Further, molecules of the invention comprising variant Fc regions may beconjugated to a therapeutic agent or a drug moiety that modifies a givenbiological response. Therapeutic agents or drug moieties are not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin (i.e., PE-40), ordiphtheria toxin, ricin, gelonin, and pokeweed antiviral protein, aprotein such as tumor necrosis factor, interferons including, but notlimited to, α-interferon (IFN-α), β-interferon (IFN-β), nerve growthfactor (NGF), platelet derived growth factor (PDGF), tissue plasminogenactivator (TPA), an apoptotic agent (e.g., TNF-α, TNF-β, AIM I asdisclosed in PCT Publication No. WO 97/33899), AIM II (see, PCTPublication No. WO 97/34911), Fas Ligand (Takahashi et al., J. Immunol.,6:1567-1574, 1994), and VEGI (PCT Publication No. WO 99/23105), athrombotic agent or an anti-angiogenic agent (e.g., angiostatin orendostatin), or a biological response modifier such as, for example, alymphokine (e.g., interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor(“GM-CSF”), and granulocyte colony stimulating factor (“G-CSF”),macrophage colony stimulating factor, (“M-CSF”), or a growth factor(e.g., growth hormone (“GH”); proteases, or ribonucleases.

Molecules of the invention can be fused to marker sequences, such as apeptide to facilitate purification. In preferred embodiments, the markeramino acid sequence is a hexa-histidine peptide, such as the tagprovided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,Calif., 91311), among others, many of which are commercially available.As described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA,86:821-824, for instance, hexa-histidine provides for convenientpurification of the fusion protein. Other peptide tags useful forpurification include, but are not limited to, the hemagglutinin “HA”tag, which corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson et al., Cell, 37:767 1984) and the “flag”tag (Knappik et al., Biotechniques, 17(4):754-761, 1994).

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of molecules of the invention (e.g.,antibodies with higher affinities and lower dissociation rates). See,generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252;and 5,837,458, and Patten et al., 1997, Curr. Opinion Biotechnol.8:724-33; Harayama, 1998, Trends Biotechnol. 16:76; Hansson, et al.,1999, J. Mol. Biol. 287:265; and Lorenzo and Blasco, 1998, BioTechniques24:308 (each of these patents and publications are hereby incorporatedby reference in its entirety). Molecules of the invention comprisingvariant Fc regions, or the nucleic acids encoding the molecules of theinvention, may be further altered by being subjected to randommutagenesis by error-prone PCR, random nucleotide insertion or othermethods prior to recombination. One or more portions of a polynucleotideencoding a molecule of the invention, may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

The present invention also encompasses molecules of the inventioncomprising variant Fc regions conjugated to a diagnostic or therapeuticagent or any other molecule for which serum half-life is desired to beincreased and/or targeted to a particular subset of cells. The moleculesof the invention can be used diagnostically to, for example, monitor thedevelopment or progression of a disease, disorder or infection as partof a clinical testing procedure to, e.g., determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling themolecules of the invention to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals, and nonradioactiveparamagnetic metal ions. The detectable substance may be coupled orconjugated either directly to the molecules of the invention orindirectly, through an intermediate (such as, for example, a linkerknown in the art) using techniques known in the art. See, for example,U.S. Pat. No. 4,741,900 for metal ions which can be conjugated toantibodies for use as diagnostics according to the present invention.Such diagnosis and detection can be accomplished by coupling themolecules of the invention to detectable substances including, but notlimited to, various enzymes, enzymes including, but not limited to,horseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; prosthetic group complexes such as, but notlimited to, streptavidin/biotin and avidin/biotin; fluorescent materialssuch as, but not limited to, umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; luminescent material such as, but not limitedto, luminol; bioluminescent materials such as, but not limited to,luciferase, luciferin, and aequorin; radioactive material such as, butnot limited to, bismuth (²¹³Bi), carbon (¹⁴C), chromium (⁵¹Cr), cobalt(⁵⁷Co), fluorine (¹⁸F), gadolinium (¹⁵³Gd, 159 Gd), gallium (⁶⁸Ga,⁶⁷Ga), germanium (⁶⁸Ge), holmium (¹⁶⁶Ho), indium (¹¹⁵In, ¹¹³In, ¹¹²In,111In), iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), lanthanium (¹⁴⁰La), lutetium(¹⁷⁷Lu), manganese (⁵⁴Mn), molybdenum (⁹⁹Mo), palladium (¹⁰³Pd),phosphorous (³²P), praseodymium (¹⁴²Pr), promethium (¹⁴⁹Pm), rhenium(¹⁸⁶Re, ¹⁸⁸Re), rhodium (¹⁰⁵Rh), ruthemium (⁹⁷Ru), samarium (¹⁵³Sm),scandium (⁴⁷Sc), selenium (⁷⁵Se), strontium (⁸⁵Sr), sulfur (³⁵S),technetium (⁹⁹Tc), thallium (²⁰¹Ti), tin (¹¹³Sn, ¹¹⁷Sn), tritium (³H),xenon (¹³³Xe), ytterbium (¹⁶⁹Yb, ¹⁷⁵Yb), yttrium (⁹⁰Y), zinc (⁶⁵Zn);positron emitting metals using various positron emission tomographies,and nonradioactive paramagnetic metal ions.

Molecules of the invention comprising a variant Fc region may beconjugated to a therapeutic moiety such as a cytotoxin (e.g., acytostatic or cytocidal agent), a therapeutic agent or a radioactiveelement (e.g., alpha-emitters, gamma-emitters, etc.). Cytotoxins orcytotoxic agents include any agent that is detrimental to cells.Examples include paclitaxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents include,but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines(e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, andanthramycin (AMC), and anti-mitotic agents (e.g., vincristine andvinblastine).

Moreover, a molecule of the invention can be conjugated to therapeuticmoieties such as a radioactive materials or macrocyclic chelators usefulfor conjugating radiometal ions (see above for examples of radioactivematerials). In certain embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) whichcan be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.,1998, Clin Cancer Res. 4:2483-90; Peterson et al., 1999, Bioconjug.Chem. 10:553; and Zimmerman et al., 1999, Nucl. Med. Biol. 26:943-50each of which is incorporated herein by reference in their entireties.

Techniques for conjugating such therapeutic moieties to antibodies arewell known; see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), 1985, pp. 243-56, Alan R.Liss, Inc.); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), 1987, pp.623-53, Marcel Dekker, Inc.); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), 1985, pp.475-506); “Analysis, Results, And Future Prospective Of The TherapeuticUse Of Radiolabeled Antibody In Cancer Therapy”, in MonoclonalAntibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),1985, pp. 303-16, Academic Press; and Thorpe et al., Immunol. Rev.,62:119-58, 1982.

In one embodiment, where the molecule of the invention is an antibodycomprising a variant Fc region, it can be administered with or without atherapeutic moiety conjugated to it, administered alone, or incombination with cytotoxic factor(s) and/or cytokine(s) for use as atherapeutic treatment. Alternatively, an antibody of the invention canbe conjugated to a second antibody to form an antibody heteroconjugateas described by Segal in U.S. Pat. No. 4,676,980, which is incorporatedherein by reference in its entirety. Antibodies of the invention mayalso be attached to solid supports, which are particularly useful forimmunoassays or purification of the target antigen. Such solid supportsinclude, but are not limited to, glass, cellulose, polyacrylamide,nylon, polystyrene, polyvinyl chloride or polypropylene.

5.2 Screening of Molecules with Variant Fc Regions for EffectorFunction, Enhanced FcγRIII Binding and Characterization of Same

The affinities and binding properties of the molecules of the inventionfor an FcγR are initially determined using in vitro assays (biochemicalor immunological based assays) known in the art for determining Fc-FcγRinteractions, i.e., specific binding of an Fc region to an FcγRincluding but not limited to ELISA assay, surface plasmon resonanceassay, immunoprecipitation assays. Preferably, the binding properties ofthe molecules of the invention are also characterized by in vitrofunctional assays for determining one or more FcγR mediator effectorcell functions. In most preferred embodiments, the antibodies of theinvention have similar binding properties in in vivo models (such asthose described and disclosed herein) as those in in vitro based assays.However, the present invention does not exclude molecules of theinvention that do not exhibit the desired phenotype in in vitro basedassays but do exhibit the desired phenotype in vivo.

In preferred embodiments, screening and identifying molecules comprisingvariant Fc regions with altered FcγR affinities (e.g., enhanced FcγRIIIAaffinity) are done using the yeast display technology as describedherein in combination with one or more biochemical based assays,preferably in a high throughput manner. In some embodiments, screeningand identifying molecules comprising variant Fc regions with alteredFcγR affinities (e.g., enhanced FcγRIIIA affinity) are done using theyeast display technology as described herein in combination with one ormore functional based assays, preferably in a high throughput manner.The functional based assays can be any assay known in the art forcharacterizing one or more FcγR mediated effector cell functions such asthose described herein in Section 5.2.7. Non-limiting examples ofeffector cell functions that can be used in accordance with the methodsof the invention, include but are not limited to, antibody-dependentcell mediated cytotoxicity (ADCC), antibody-dependent phagocytosis,phagocytosis, opsonization, opsonophagocytosis, cell binding, rosetting,C1q binding, and complement dependent cell mediated cytotoxicity.

The term “specific binding” of an Fc region to an FcγR refers to aninteraction of the Fc region and a particular FcγR which has an affinityconstant of at least about 150 nM, in the case of monomeric FcγRIIIA andat least about 60 nM in the case of dimeric FcγRIIB as determined using,for example, an ELISA or surface plasmon resonance assay (e.g., aBIAcore™). The affinity constant of an Fc region for monomeric FcγRIIIAmay be 150 nM, 200 nM or 300 nM. The affinity constant of an Fc regionfor dimeric FcγRIIB may be 60 nM, 80 nM, 90 nM, or 100 nM. DimericFcγRIIB for use in the methods of the invention may be generated usingmethods known to one skilled in the art. Typically, the extracellularregion of FcγRIIB is covalently linked to a heterologous polypeptidewhich is capable of dimerization, so that the resulting fusion proteinis a dimer, e.g., see, U.S. Application No. 60/439,709 filed on Jan. 13,2003 (Attorney Docket No. 11183-005-888), which is incorporated hereinby reference in its entirety. A specific interaction generally is stableunder physiological conditions, including, for example, conditions thatoccur in a living individual such as a human or other vertebrate orinvertebrate, as well as conditions that occur in a cell culture suchconditions as used for maintaining and culturing mammalian cells orcells from another vertebrate organism or an invertebrate organism.

In a specific embodiment, screening for and identifying moleculescomprising variant Fc regions and altered FcγR affinities comprise:displaying the molecule comprising a variant Fc region on the yeastsurface; and characterizing the binding of the molecule comprising thevariant Fc region to a FcγR (one or more), using a biochemical assay fordetermining Fc-FcγR interaction, preferably, an ELISA based assay. Oncethe molecule comprising a variant Fc region has been characterized forits interaction with one or more FcγRs and determined to have an alteredaffinity for one or more FcγRs, by at least one biochemical based assay,e.g., an ELISA assay, the molecule maybe engineered into a completeimmunoglobulin, such as a molecule, using standard recombinant DNAtechnology methods known in the art, and the immunoglobulin comprisingthe variant Fc region expressed in mammalian cells for furtherbiochemical characterization. The immunoglobulin into which a variant Fcregion of the invention is introduced (e.g., replacing the Fc region ofthe immunoglobulin) can be any immunoglobulin including, but not limitedto, polyclonal antibodies, monoclonal antibodies, bispecific antibodies,multi-specific antibodies, humanized antibodies, and chimericantibodies. In preferred embodiments, a variant Fc region is introducedinto an immunoglobulin specific for a cell surface receptor, a tumorantigen, or a cancer antigen. The immunoglobulin into which a variant Fcregion of the invention is introduced may specifically bind a cancer ortumor antigen for example, including, but not limited to, KS 1/4pan-carcinoma antigen (Perez and Walker, 1990, J. Immunol. 142:3662-3667; Bumal, 1988, Hybridoma 7(4): 407-415), ovarian carcinomaantigen (CA125) (Yu et al., 1991, Cancer Res. 51(2): 468-475), prostaticacid phosphate (Tailor et al., 1990, Nucl. Acids Res. 18(16): 4928),prostate specific antigen (Henttu and Vihko, 1989, Biochem. Biophys.Res. Comm. 160(2): 903-910; Israeli et al., 1993, Cancer Res. 53:227-230), melanoma-associated antigen p97 (Estin et al., 1989, J. Natl.Cancer Instil. 81(6): 445-446), melanoma antigen gp75 (Vijayasardahl etal., 1990, J. Exp. Med. 171(4): 1375-1380), high molecular weightmelanoma antigen (HMW-MAA) (Natali et al., 1987, Cancer 59: 55-63;Mittelman et al., 1990, J. Clin. Invest. 86: 2136-2144), prostatespecific membrane antigen, carcinoembryonic antigen (CEA) (Foon et al.,1994, Proc. Am. Soc. Clin. Oncol. 13: 294), polymorphic epithelial mucinantigen, human milk fat globule antigen, colorectal tumor-associatedantigens such as: CEA, TAG-72 (Yokata et al., 1992, Cancer Res. 52:3402-3408), C017-1A (Ragnhammar et al., 1993, Int. J. Cancer 53:751-758); GICA 19-9 (Herlyn et al., 1982, J. Clin. Immunol. 2: 135),CTA-1 and LEA, Burkitt's lymphoma antigen-38.13, CD19 (Ghetie et al.,1994, Blood 83: 1329-1336), human B-lymphoma antigen-CD20 (Reff et al.,1994, Blood 83:435-445), CD33 (Sgouros et al., 1993, J. Nucl. Med.34:422-430), melanoma specific antigens such as ganglioside GD2 (Salehet al., 1993, J. Immunol., 151, 3390-3398), ganglioside GD3 (Shitara etal., 1993, Cancer Immunol. Immunother. 36:373-380), ganglioside GM2(Livingston et al., 1994, J. Clin. Oncol. 12: 1036-1044), gangliosideGM3 (Hoon et al., 1993, Cancer Res. 53: 5244-5250), tumor-specifictransplantation type of cell-surface antigen (TSTA) such asvirally-induced tumor antigens including T-antigen DNA tumor viruses andEnvelope antigens of RNA tumor viruses, oncofetalantigen-alpha-fetoprotein such as CEA of colon, bladder tumor oncofetalantigen (Hellstrom et al., 1985, Cancer. Res. 45:2210-2188),differentiation antigen such as human lung carcinoma antigen L6, L20(Hellstrom et al., 1986, Cancer Res. 46: 3917-3923), antigens offibrosarcoma, human leukemia T cell antigen-Gp37(Bhattacharya-Chatterjee et al., 1988, J. of Immun. 141:1398-1403),neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR(Epidermal growth factor receptor), HER2 antigen (p185^(HER2)),polymorphic epithelial mucin (PEM) (Hilkens et al., 1992, Trends in Bio.Chem. Sci. 17:359), malignant human lymphocyte antigen-APO-1 (Bernhardet al., 1989, Science 245: 301-304), differentiation antigen (Feizi,1985, Nature 314: 53-57) such as I antigen found in fetal erythrocytes,primary endoderm I antigen found in adult erythrocytes, preimplantationembryos, I(Ma) found in gastric adenocarcinomas, M18, M39 found inbreast epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9, Myl,VIM-D5, D₁56-22 found in colorectal cancer, TRA-1-85 (blood group H),C14 found in colonic adenocarcinoma, F3 found in lung adenocarcinoma,AH6 found in gastric cancer, Y hapten, Le^(y) found in embryonalcarcinoma cells, TL5 (blood group A), EGF receptor found in A431 cells,E₁ series (blood group B) found in pancreatic cancer, FC10.2 found inembryonal carcinoma cells, gastric adenocarcinoma antigen, CO-514 (bloodgroup Le^(a)) found in Adenocarcinoma, NS-10 found in adenocarcinomas,CO-43 (blood group Le^(b)), G49 found in EGF receptor of A431 cells, MH2(blood group ALe^(b)/Le^(y)) found in colonic adenocarcinoma, 19.9 foundin colon cancer, gastric cancer mucins, T₅A₇ found in myeloid cells, R₂₄found in melanoma, 4.2, G_(D3), D1.1, OFA-1, G_(M2), OFA-2, G_(D2), andM1:22:25:8 found in embryonal carcinoma cells, and SSEA-3 and SSEA-4found in 4 to 8-cell stage embryos. In one embodiment, the antigen is aT cell receptor derived peptide from a Cutaneous T cell Lymphoma (see,Edelson, 1998, The Cancer Journal 4:62).

In some embodiments, a variant Fc region of the invention is introducedinto an anti-fluoresceine monoclonal antibody, 4-4-20 (Kranz et al.,1982, J. Biol. Chem. 257(12): 6987-6995; which is incorporated herein byreference in its entirety). In other embodiments, a variant Fc region ofthe invention is introduced into a mouse-human chimeric anti-CD20monoclonal antibody, 2H7, which recognizes the CD20 cell surfacephosphoprotein on B cells (Liu et al., 1987, Journal of Immunology, 139:3521-6; which is incorporated herein by reference in its entirety). Inother embodiments, the monoclonal antibody is not an anti-CD20 antibody.In yet other embodiments, a variant Fc region of the invention isintroduced into a humanized antibody (Ab4D5) against the human epidermalgrowth factor receptor 2 (p185 HER2) as described by Carter et al.(1992, Proc. Natl. Acad. Sci. USA 89: 4285-9; which is incorporatedherein by reference in its entirety). In yet other embodiments, avariant Fc region of the invention is introduced into a humanizedanti-TAG72 antibody (CC49) (Sha et al., 1994 Cancer Biother. 9(4):341-9; which is incorporated herein by reference in its entirety). Inother embodiments, a variant Fc region of the invention is introducedinto RITUXAN™ (humanized anti-CD20 antibody; rituximab) (InternationalPatent Publication No. WO 02/096948; which is incorporated herein byreference in its entirety) which is used for treating lymphomas.

In another specific embodiment, the invention encompasses engineering ananti-FcγRIIB antibody including but not limited to any of the antibodiesdisclosed in U.S. Provisional Application No. 60/403,266 filed on Aug.12, 2002; U.S. application Ser. No. 10/643,857 filed on Aug. 14, 2003(having Attorney Docket No. 011183-010-999); the U.S. ProvisionalApplication No. 60/562,804 (having Attorney Docket No. 011183-014-888)that was filed on Apr. 16, 2004; U.S. Provisional Application No.60/569,882 (having Attorney Docket No. 011183-013-888) that was filed onMay 10, 2004 and U.S. Provisional Application Nos. 60/582,044,60/582,045, and 60/582,043, having Attorney Docket Nos. 011183-016-888,011183-017-888, and 011183-018-888, respectively, each of which wasfiled on Jun. 21, 2004, by modification (e.g., substitution, insertion,deletion) of at least one amino acid residue which modificationincreases the affinity of the Fc region for FcγRIIIA and/or FcγRIIA.Examples of anti-FcγRIIB antibodies that may be engineered in accordancewith the methods of the invention are 2B6 monoclonal antibody havingATCC accession number PTA-4591 and 3H7 having ATCC accession numberPTA-4592, 1D5 monoclonal antibody having ATCC accession number PTA-5958,1F2 monoclonal antibody having ATCC accession number PTA-5959, 2D11monoclonal antibody having ATCC accession number PTA-5960, 2E1monoclonal antibody having ATCC accession number PTA-5961 and 2H9monoclonal antibody having ATCC accession number PTA-5962 (all depositedat 10801 University Boulevard, Manassas, Va. 02209-2011 under the termsof the Budapest Treaty on the International Recognition of the Depositof Microorganisms for the Purposes of Patent Procedure), which areincorporated herein by reference. In another specific embodiment,modification of the anti-FcγRIIB antibody may also further decrease theaffinity of the Fc region for FcγRIIB. In yet another specificembodiment, the engineered anti-FcγRIIB antibody may further have anenhanced effector function as determined by standard assays known in theart and disclosed and exemplified herein. In some embodiments, a variantFc region of the invention is introduced into a therapeutic monoclonalantibody specific for a cancer antigen or cell surface receptorincluding but not limited to, ERBITUX™ anti-EGFR antibody (also known asIMC-C225) (ImClone Systems Inc.), a chimerized monoclonal antibodyagainst EGFR; HERCEPTIN® anti-HER2 antibody (Trastuzumab) (Genentech,CA) which is a humanized anti-HER2 monoclonal antibody for the treatmentof patients with metastatic breast cancer; REOPRO® anti-glycoproteinIIb/IIIa receptor antibody (abciximab) (Centocor) which is ananti-glycoprotein IIb/IIIa receptor on the platelets for the preventionof clot formation; ZENAPAX® anti-CD25 antibody (daclizumab) (RochePharmaceuticals, Switzerland) which is an immunosuppressive, humanizedanti-CD25 monoclonal antibody for the prevention of acute renalallograft rejection. Other examples are a humanized anti-CD18 F(ab′)₂(Genentech); CDP860 which is a humanized anti-CD18 F(ab′)₂ (Celltech,UK); PRO542 which is an anti-HIV gp120 antibody fused with CD4(Progenics/Genzyme Transgenics); C14 which is an anti-CD14 antibody(ICOS Pharm); a humanized anti-VEGF IgG1 antibody (Genentech); OVAREX™anti-CA 125 antibody which is a murine anti-CA 125 antibody (Altarex);PANOREX™ anti-17-IA antibody which is a murine anti-17-IA cell surfaceantigen IgG2a antibody (Glaxo Wellcome/Centocor); IMC-C225 which is achimeric anti-EGFR IgG antibody (ImClone System); VITAXIN™ anti-αVβ3integrin antibody which is a humanized anti-αVβ3 integrin antibody(Applied Molecular Evolution/MedImmune); CAMPATH® anti CD52 antibody1H/LDP-03 which is a humanized anti CD52 IgG1 antibody (Leukosite);SMART 195™ anti-CD33 antibody which is a humanized anti-CD33 IgGantibody (Protein Design Lab/Kanebo); RITUXAN™ (rituximab) anti-CD20antibody which is a chimeric anti-CD20 IgG1 antibody (IDECPharm/Genentech, Roche/Zettyaku); LYMPHOCIDE™ anti-CD22 antibody whichis a humanized anti-CD22 IgG antibody (Immunomedics); SMART ID10™anti-HLA antibody which is a humanized anti-HLA antibody (Protein DesignLab); ONCOLYM™ (Lym-1) anti-HLA DR antibody is a radiolabelled murineanti-HLA DR antibody (Techniclone); anti-CD11a is a humanized IgG1antibody (Genetech/Xoma); ICM3™ anti-ICAM3 is a humanized anti-ICAM3antibody (ICOS Pharm); IDEC-114™ anti-CD80 antibody is a primatizedanti-CD80 antibody (IDEC Pharm/Mitsubishi); ZEVALIN™ anti-CD20 antibodyis a radiolabelled murine anti-CD20 antibody (IDEC/Schering AG);IDEC-131™ anti-CD40L antibody is a humanized anti-CD40L antibody(IDEC/Eisai); IDEC-151™ anti-CD4 antibody is a primatized anti-CD4antibody (IDEC); IDEC-152™ anti-CD23 antibody is a primatized anti-CD23antibody (IDEC/Seikagaku); SMART anti-CD3™ anti-CD3 antibody is ahumanized anti-CD3 IgG (Protein Design Lab); 5G1.1 is a humanizedanti-complement factor 5 (C5) antibody (Alexion Pharm); IDEC-151™anti-CD4 antibody is a primatized anti-CD4 IgG1 antibody (IDECPharm/SmithKline Beecham); MDX-CD4™ anti-CD4 antibody is a humananti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571™ anti-TNF-αantibody is a humanized anti-TNF-α IgG4 antibody (Celltech); LDP-02™anti-α4β7 antibody is a humanized anti-α4β7 antibody(LeukoSite/Genentech); OrthoClone OKT4A™ anti-CD4 antibody is ahumanized anti-CD4 IgG antibody (Ortho Biotech); ANTOVA™ anti-CD40antibody is a humanized anti-CD40L IgG antibody (Biogen); ANTEGREN™anti-VLA-4 antibody is a humanized anti-VLA-4 IgG antibody (Elan);MDX-33™ anti-CD64 antibody is a human anti-CD64 (FcγR) antibody(Medarex/Centeon); rhuMab-E25™ anti-IgE antibody is a humanized anti-IgEIgG1 antibody (Genentech/Norvartis/Tanox Biosystems); IDEC-152™anti-CD64 antibody is a primatized anti-CD23 antibody (IDEC Pharm);ABX-CBL™ anti-CD147 antibody is a murine anti CD-147 IgM antibody(Abgenix); BTI-322™ anti-CD2 antibody is a rat anti-CD2 IgG antibody(Medimmune/Bio Transplant); Orthoclone/OKT3™ anti-CD3 antibody is amurine anti-CD3 IgG2a antibody (ortho Biotech); SIMULECT™ anti-CD25antibody is a chimeric anti-CD25 IgG1 antibody (Novartis Pharm); LDP-01™anti-β₂-integrin antibody is a humanized anti-β₂-integrin IgG antibody(LeukoSite); Anti-LFA-1™ anti-CD18 antibody is a murine anti CD18F(ab′)₂ (Pasteur-Merieux/Immunotech); CAT-152™ anti-TGF-β₂ antibody is ahuman anti-TGF-β₂ antibody (Cambridge Ab Tech); and Corsevin M™anti-Factor VII antibody is a chimeric anti-Factor VII antibody(Centocor). In certain embodiments, the antibody is not RITUXAN™anti-CD20 antibody.

The variant Fc regions of the invention, preferably in the context of animmunoglobulin, can be further characterized using one or morebiochemical assays and/or one or more functional assays, preferably in ahigh throughput manner. In some alternate embodiments, the variant Fcregions of the inventions are not introduced into an immunoglobulin andare further characterized using one or more biochemical based assaysand/or one or more functional assays, preferably in a high throughputmanner. The one or more biochemical assays can be any assay known in theart for identifying immunoglobulin-antigen or Fc-FcγR interactions,including, but not limited to, an ELISA assay, and surface plasmonresonance-based assay, e.g., BIAcore assay, for determining the kineticparameters of immunoglobulin-antigen or Fc-FcγR interaction.Characterization of target antigen binding affinity or assessment oftarget antigen density on a cell surface may be assessed by methods wellknown in the art such as Scatchard analysis or by the use of kits as permanufacturer's instructions, such as Quantum™ Simply Cellular® (BangsLaboratories, Inc., Fishers, Ind.). The one or more functional assayscan be any assay known in the art for characterizing one or more FcγRmediated effector cell function as known to one skilled in the art ordescribed herein. In specific embodiments, the immunoglobulinscomprising the variant Fc regions are assayed in an ELISA assay forbinding to one or more FcγRs, e.g., FcγRIIIA, FcγRIIA, FcγRIIA; followedby one or more ADCC assays. In some embodiments, the immunoglobulinscomprising the variant Fc regions are assayed further using a surfaceplasmon resonance-based assay, e.g., BIAcore. Surface plasmonresonance-based assays are well known in the art, and are furtherdiscussed in Section 5.2.7, and exemplified herein in Example 6.8.

An exemplary high throughput assay for characterizing immunoglobulinscomprising variant Fc regions may comprise: introducing a variant Fcregion of the invention, e.g., by standard recombinant DNA technologymethods, in a 4-4-20 antibody; characterizing the specific binding ofthe 4-4-20 antibody comprising the variant Fc region to an FcγR (e.g.,FcγRIIIA, FcγRIIB) in an ELISA assay; characterizing the 4-4-20 antibodycomprising the variant Fc region in an ADCC assay (using methodsdisclosed herein) wherein the target cells are opsonized with the 4-4-20antibody comprising the variant Fc region; the variant Fc region maythen be cloned into a second immunoglobulin, e.g., 4D5, 2H7, and thatsecond immunoglobulin characterized in an ADCC assay, wherein the targetcells are opsonized with the second antibody comprising the variant Fcregion. The second antibody comprising the variant Fc region is thenfurther analyzed using an ELISA-based assay to confirm the specificbinding to an FcγR.

Preferably, a variant Fc region of the invention binds FcγRIIIA and/orFcγRIIA with a higher affinity than a wild type Fc region as determinedin an ELISA assay. Most preferably, a variant Fc region of the inventionbinds FcγRIIIA and/or FcγRIIA with a higher affinity and binds FcγRIIBwith a lower affinity than a wild type Fc region as determined in anELISA assay. In some embodiments, the variant Fc region binds FcγRIIIAand/or FcγRIIA with at least 2-fold higher, at least 4-fold higher, morepreferably at least 6-fold higher, most preferably at least 8 to 10-foldhigher affinity than a wild type Fc region binds FcγRIIIA and/or FcγRIIAand binds FcγRIIB with at least 2-fold lower, at least 4-fold lower,more preferably at least 6-fold lower, most preferably at least 8 to10-fold lower affinity than a wild type Fc region binds FcγRIIB asdetermined in an ELISA assay.

The immunoglobulin comprising the variant Fc regions may be analyzed atany point using a surface plasmon based resonance based assay, e.g.,BIAcore, for defining the kinetic parameters of the Fc-FcγR interaction,using methods disclosed herein and known to those of skill in the art.Preferably, the Kd of a variant Fc region of the invention for bindingto a monomeric FcγRIIIA and/or FcγRIIA as determined by BIAcore analysisis about 100 nM, preferably about 70 nM, most preferably about 40 nM.;and the Kd of the variant Fc region of the invention for binding adimeric FcγRIIB is about 80 nM, about 100 nM, more preferably about 200nM.

In most preferred embodiments, the immunoglobulin comprising the variantFc regions is further characterized in an animal model for interactionwith an FcγR. Preferred animal models for use in the methods of theinvention are, for example, transgenic mice expressing human FcγRs,e.g., any mouse model described in U.S. Pat. Nos. 5,877,397, and6,676,927 which are incorporated herein by reference in their entirety.Transgenic mice for use in the methods of the invention include, but arenot limited to, nude knockout FcγRIIIA mice carrying human FcγRIIIA;nude knockout FcγRIIIA mice carrying human FcγRIIA; nude knockoutFcγRIIIA mice carrying human FcγRIIB and human FcγRIIIA; nude knockoutFcγRIIIA mice carrying human FcγRIIB and human FcγRIIA; nude knockoutFcγRIIIA and FcγRIIA mice carrying human FcγRIIIA and FcγRIIA and nudeknockout FcγRIIIA, FcγRIIA and FcγRIIB mice carrying human FcγRIIIA,FcγRIIA and FcγRIIB.

5.2.1 Design Strategies

The present invention encompasses engineering methods to generate Fcvariants including but not limited to computational design strategies,library generation methods, and experimental production and screeningmethods. These strategies may be applied individually or in variouscombinations to engineer the Fc variants of the instant invention.

In most preferred embodiments, the engineering methods of the inventioncomprise methods in which amino acids at the interface between an Fcregion and the Fc ligand are not modified. Fc ligands include but arenot limited to FcγRs, C1q, FcRn, C3, mannose receptor, protein A,protein G, mannose receptor, and undiscovered molecules that bind Fc.Amino acids at the interface between an Fc region and an Fc ligand isdefined as those amino acids that make a direct and/or indirect contactbetween the Fc region and the ligand, play a structural role indetermining the conformation of the interface, or are within at least 3angstroms, preferably at least 2 angstroms of each other as determinedby structural analysis, such as x-ray crystallography and molecularmodeling The amino acids at the interface between an Fc region and an Fcligand include those amino acids that make a direct contact with an FcγRbased on crystallographic and structural analysis of Fc-FcγRinteractions such as those disclosed by Sondermann et al., (2000,Nature, 406: 267-273; which is incorporated herein by reference in itsentirety). Examples of positions within the Fc region that make a directcontact with FcγR are amino acids 234-239 (hinge region), amino acids265-269 (B/C loop), amino acids 297-299 (C′/E loop), and amino acids327-332 (F/G) loop. In some embodiments, the molecules of the inventioncomprising variant Fc regions comprise modification of at least oneresidue that does not make a direct contact with an FcγR based onstructural and crystallographic analysis, e.g., is not within theFc-FcγR binding site.

Preferably, the engineering methods of the invention do not modify anyof the amino acids as identified by Shields et al., which are located inthe CH2 domain of an Fc region proximal to the hinge region, e.g.,Leu234-Pro238; Ala327, Pro329, and affect binding of an Fc region to allhuman FcγRs.

In other embodiments, the invention encompasses Fc variants with alteredFcγR affinities and/or altered effector functions, such that the Fcvariant does not have an amino acid modification at a position at theinterface between an Fc region and the Fc ligand. Preferably, such Fcvariants in combination with one or more other amino acid modificationswhich are at the interface between an Fc region and the Fc ligand have afurther impact on the particular altered property, e.g. altered FcγRaffinity. Modifying amino acids at the interface between Fc and an Fcligand may be done using methods known in the art, for example based onstructural analysis of Fc-ligand complexes. For example but not by wayof limitation by exploring energetically favorable substitutions at Fcpositions that impact the binding interface, variants can be engineeredthat sample new interface conformations, some of which may improvebinding to the Fc ligand, some of which may reduce Fc ligand binding,and some of which may have other favorable properties. Such newinterface conformations could be the result of, for example, directinteraction with Fc ligand residues that form the interface, or indirecteffects caused by the amino acid modifications such as perturbation ofside chain or backbone conformations

The invention encompasses engineering Fc variants comprising any of theamino acid modifications disclosed herein in combination with othermodifications in which the conformation of the Fc carbohydrate atposition 297 is altered. The invention encompasses conformational andcompositional changes in the N297 carbohydrate that result in a desiredproperty, for example increased or reduced affinity for an FcγR. Suchmodifications may further enhance the phenotype of the original aminoacid modification of the Fc variants of the invention. Although notintending to be bound by a particular mechanism of actions such astrategy is supported by the observation that the carbohydrate structureand conformation dramatically affect Fc-FcγR and Fc/C1q binding (Umahaet al., 1999, Nat Biotechnol 17:176-180; Davies et al., 2001, BiotechnolBioeng 74:288-294; Mimura et al., 2001, J Biol Chem 276:45539; Radaev etal., 2001, J Biol Chem 276:16478-16483; Shields et al. 2002, J Biol Chem277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473).

Another design strategy for generating Fc variants in accordance withthe invention is provided in which the Fc region is reengineered toeliminate the structural and functional dependence on glycosylation.This design strategy involves the optimization of Fc structure,stability, solubility, and/or Fc function (for example affinity of Fcfor one or more Fc ligands) in the absence of the N297 carbohydrate. Inone approach, positions that are exposed to solvent in the absence ofglycosylation are engineered such that they are stable, structurallyconsistent with Fc structure, and have no tendency to aggregate.Approaches for optimizing aglycosylated Fc may involve but are notlimited to designing amino acid modifications that enhance aglycosylatedFc stability and/or solubility by incorporating polar and/or chargedresidues that face inward towards the Cg2-Cg2 dimer axis, and bydesigning amino acid modifications that directly enhance theaglycosylated Fc-FcγR interface or the interface of aglycosylated Fcwith some other Fc ligand.

The Fc variants of the present invention may be combined with other Fcmodifications, including but not limited to modifications that enhanceeffector function. The invention encompasses combining an Fc variant ofthe invention with other Fc modifications to provide additive,synergistic, or novel properties in antibodies or Fc fusions. Suchmodifications may be in the CH1, CH2, or CH3 domains or a combinationthereof. Preferably the Fc variants of the invention enhance theproperty of the modification with which they are combined. For example,if an Fc variant of the invention is combined with a mutant known tobind FcγRIIIA with a higher affinity than a comparable moleculecomprising a wild type Fc region; the combination with a mutant of theinvention results in a greater fold enhancement in FcγRIIIA affinity.

In one embodiment, the Fc variants of the present invention may becombined with other known Fc variants such as those disclosed in Duncanet al, 1988, Nature 332:563-564; Lund et al., 1991, J. Immunol.147:2657-2662; Lund et al, 1992, Mol Immunol 29:53-59; Alegre et al,1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl.Acad Sci USA 92:11980-11984; Jefferis et al, 1995, Immunol Lett.44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et al, 1996,Immunol Lett 54:101-104; Lund et al, 1996, J Immunol 157:49634969;Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogie et al, 2000, JImmunol 164:41784184; Reddy et al, 2000, J Immunol 164:1925-1933; Xu etal., 2000, Cell Immunol 200:16-26; Idusogie et al, 2001, J Immunol166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferiset al, 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem SocTrans 30:487-490); U.S. Pat. No. 5,624,821; U.S. Pat. No. 5,885,573;U.S. Pat. No. 6,194,551; PCT WO 00/42072; PCT WO 99/58572; each of whichis incorporated herein by reference in its entirety.

5.2.2 FcγR-Fc Binding Assay

An FcγR-Fc binding assay was developed for determining the binding ofthe molecules of the invention comprising variant Fc regions to FcγR,which allowed detection and quantization of the interaction, despite theinherently weak affinity of the receptor for its ligand, e.g., in themicro molar range for FcγRIIB and FcγRIIIA. The method involves theformation of an FcγR complex that has an improved avidity for an Fcregion, relative to an uncompleted FcγR. According to the invention, thepreferred molecular complex is a tetrameric immune complex, comprising:(a) the soluble region of FcγR (e.g., the soluble region of FcγRIIIA,FcγRIIA or FcγRIIB); (b) a biotinylated 15 amino acid AVITAG™ peptidesequence operably linked to the C-terminus of the soluble region of FcγR(e.g., the soluble region of FcγRIIIA, FcγRIIA or FcγRIIB); and (c)streptavidin-phycoerythrin (SA-PE); in a molar ratio to form atetrameric FcγR complex (preferably in a 5:1 molar ratio). According toa preferred embodiment of the invention, the fusion protein isbiotinylated enzymatically, using for example, the E. coli Bir A enzyme,a biotin ligase which specifically biotinylates a lysine residue in the15 amino acid AVITAG™ peptide sequence. In a specific embodiment of theinvention, 85% of the fusion protein is biotinylated, as determined bystandard methods known to those skilled in the art, including but notlimited to streptavidin shift assay. According to preferred embodimentsof the invention, the biotinylated soluble FcγR proteins are mixed withSA-PE in a 1× SA-PE:5× biotinylated soluble FcγR molar ratio to form atetrameric FcγR complex.

In a preferred embodiment of the invention, molecules comprising Fcregions bind the tetrameric FcγR complexes, formed according to themethods of the invention, with at least an 8-fold higher affinity thanthe monomeric uncomplexed FcγR. The binding of polypeptides comprisingFc regions to the tetrameric FcγR complexes may be determined usingstandard techniques known to those skilled in the art, such as forexample, fluorescence activated cell sorting (FACS), radioimmunoassays,ELISA assays, etc.

The invention encompasses the use of the immune complexes formedaccording to the methods described above, for determining thefunctionality of molecules comprising an Fc region in cell-based orcell-free assays.

As a matter of convenience, the reagents may be provided in an assaykit, i.e., a packaged combination of reagents for assaying the abilityof molecules comprising variant Fc regions to bind FcγR tetramericcomplexes. Other forms of molecular complexes for use in determiningFc-FcγR interactions are also contemplated for use in the methods of theinvention, e.g., fusion proteins formed as described in U.S. ProvisionalApplication 60/439,709, filed on Jan. 13, 2003 (Attorney Docket No.11183-005-888); which is incorporated herein by reference in itsentirety.

5.2.3 Mutagenesis and Construction of Yeast Display Libraries

Molecular interactions between the IgG Fc and Fc receptors have beenpreviously studied by both structural and genetic techniques. Thesestudies identified amino acid residues that are critical for functionalbinding of Fc to different FcγR. None of these changes have been shownto improve human FcγR mediated efficacy of therapeutic antibodies inanimal models. A complete analysis of all potential amino acid changesat these residues or other potentially important residues has not beenreported. The platform described herein has the ability to bothconstruct mutant libraries with all possible amino acid changes, screenlibraries using multiple functional assays, and finally analyzelibraries in relevant humanized animal models.

The instant invention encompasses construction of multiple librariesbased on both genetic and structural data known in the art or beingdeveloped. The method described and exemplified herein incorporatesbuilding individual libraries that contain mutants testing all 20 aminoacid changes at between 3-6 residues in the Fc region. The complete setof mutations will be assembled in all possible combinations ofmutations. The number of independent mutations generated is based on thenumber of sites being saturated during library assembly (Table 9 below).Library size will determine the choice of primary screen and thereforethe choice of vector for initial cloning steps.

TABLE 9 Number of Independent mutants based on number of targeted sites.Library # of residues # independent mutants Primary screen Small 3 orless 8000 max. ELISA Large 4-6 1.6 × 10⁵-6.4 × 10⁷ Surface display

The instant invention encompasses construction of combinatoriallibraries, focusing on a limited number of critical residues (e.g.,3-6). Using a library of randomly mutagenized IgG1 Fc and the screeningassays described and exemplified herein Fc variants will be identified.In the initial rounds, the best 5 mutations, based on both FcR bindingprofile and functional activity will be selected. It will take 20⁵individual mutants to cover all possible amino acid changes and theircombinations at five locations. A library with at least 10-fold coveragefor each mutant will be generated. In addition regions will be chosenbased on available information, e.g., crystal structure data,Mouse/Human isotype FcγR binding differences, genetic data, andadditional sites identified by mutagenesis.

The biggest disadvantage of current site directed mutagenic protocols isproduction of bias populations, over-representing variations in someregions and under-representing or completely lacking mutations inothers. The present invention overcomes this problem by generatingunbiased arrays of desirable Fc mutants using a well-developed genebuilding technology to eliminate the bias introduced in libraryconstruction by PCR based approaches such as overlapping PCR andinverted PCR. The key distinctions of the approach of the presentinvention are: 1) Employment of equimolar mix of 20 individual oligosfor every targeted codon instead of degenerated primers. This way eachamino acid is represented by a single, most used codon, whereasdegenerated primers over represent those amino acids encoded by morecodons over those encoded by fewer codons. 2) Building mutants by achain replacement approach. This insures unbiased introduction of alldesirable changes into the final product.

An exemplary protocol comprises of the following steps: 1)phosphorylated oligos, representing desirable changes at one or severallocations, all complementary to the same strand, added to the templatealong with a thermostable, 5′>3′ exonuclease deficient, DNA polymeraseand ligase (FIG. 26 a). 2) assembled mix undergoes a number ofpolymerization/ligation cycles, sufficient to generate desirable amountof product. Use of a 5′>3′ exonuclease deficient DNA polymerase insuresintegrity of the primer sequence and its phosphate residue, when athermostable ligase assembles individual primer-extended fragments intoa contiguous single-stranded chain. Reaction cycles can continue untilcomplete exhaustion of the oligos pool without introducing bias into thefinal product (FIG. 26 b). 3) generated pool of single-stranded mutantsis converted into double-stranded DNA by adding a reverse gene-specificprimer to the reaction (FIG. 26 1 c). 4) double-stranded product getsdigested at the end-designed restriction sites and cloned into anappropriate expression vector (FIG. 26 1 d)

To insure quality of the library, PCR amplified fragments will beanalyzed by electrophoresis to determine the length of the final PCRproducts. The reaction will be characterized as successful if >99% ofthe PCR products are of the expected length. The final library will becloned into an expression vector. A fraction of the mutant library willbe sequenced to determine the rate of mutant codon incorporation. Thenumber of fragments sequenced will be based on the number of targetsites mutated and library validation will be determined by the observedrate of mutation at targeted sites (Table 10). The rate of vectorwithout inserts should be less than 2%. The rate of mutation atnon-targeted sites should be less than 8%. Libraries containing cloneswith >90% correct inserts will allow us to maintain screening timelines.

TABLE 10 Expected rates of Mutation for Libraries Approx. rates ofmutation for library validation Targeted # of seq. Sin- Residuesreactions gle Double Triple Quad. Pent. Hex. 3 20 42% 43% 15% NA NA NA 450 29% 43% 21%  7% NA NA 5 75 18% 35% 32% 11% 4% NA 6 100 12% 20% 40%20% 6% 2%

In other embodiments, the invention the invention encompassesoverlapping or inverted PCR for construction of libraries. In order toremain unbiased, individual primers for each codon will be used ratherthan degenerative primers. A similar validation scheme as disclosedsupra will be employed.

Most preferably automated protocols will be employed for high throughputlibrary production. Automation allows for improved throughput, walk awayoperation, and an overall reduction in experimental error for tasksrequiring tedious repetitive operations. Oligo synthesis capabilities isbased on 2 Mermade DNA synthesizers (Bioautomation, Inc.) with a totaloutput capability of 575 60mer Oligos/12 hrs. Proprietary softwarehandles all aspects of design, synthesis, and storage of the finaloligonucleotides. Robotic liquid handlers will be employed to set upoligos for synthesis of full length Fc mutants and ligation reactionsfor incorporating the mutant Fcs into antibody heavy chain expressionvectors will be set up. After ligation it is estimated that it wouldtake 1 FTE ˜10 days to array the library clones and generate ˜8000minipreps, equivalent to a combinatorial library saturated at 3 sites.Subsequent to bacterial transformation a Qpix-2 clone picker robot willbe used for picking colonies into 96 deep well plates. Culture growthwill be done using a magnetic levitation stirrer, capable of incubating12 plates and resulting in dense growth in 12-16 hr at 37° C. A Qiagenminiprep robot will be used to perform DNA preps at the rate of 4 96well plates in 2.5 hrs. By overlapping tasks 5 such libraries could beconstructed in 9 months with 1 FTE.

Affinity maturation requires the assembly of a new set of combinationsof mutations, from a preselected mutant pool or members of a genefamily, which can be enriched by a selection protocol. The process isrepeated several times until the isolation of a mutant with the desiredphenotype is achieved. The disadvantage of the current enzymaticapproach, DNA shuffling, to accomplish this process is bias which can beintroduced due to specific sites within gene that are hot spots fornucleases, dominance of specific mutants in the final reassembled pooland loss of some of the original mutants in the final pool. In order toovercome this shortcoming a build-a-gene (BAG) technology will be usedto generate a highly complex library of Fc mutants containing randomamino acid changes at all potential locations that may be important forreceptor(s) binding. Sets of degenerated oligos covering specificregions of the IgG Fc will be used (See FIG. 27).

Oligos will be ˜30 nt and degenerate oligos synthesized to change one (4oligos) or two AAs (8 oligos) will be constructed. The oligos aredesigned to be overlapping with no gaps. It will take ˜200 oligos toaccommodate all single AA changes and ˜2000 to change two AAs peroligonucleotide. All 2000+ oligos will be used individually and incombinations to generate arrays of Fc mutants using the protocoloutlined above (A.20). We will use a home-written randomizer program anda robotic liquid handler for pooling selected combinations of mutant andwild type oligos. Large libraries will be cloned into vectors that willallow for screening using yeast surface display. This approach utilizesa magnetic bead selection followed by flow cytometry and has beensuccessfully applied to libraries with a complexity >10⁹ (Feldhaus etal., 2003, Nat. Biotech. 21(2): 163-170; which is incorporated herein byreference in its entirety). This limits the number of sites to test atany one pool to 7, resulting in ˜1.3×10⁹ possible mutations/pool.

To insure quality of the library PCR amplified fragments will beanalyzed by electrophoresis to determine the length of the final PCRproducts. The reaction will be characterized as successful if >99% ofthe PCR products are of the expected length. A fraction of the mutantlibrary will be sequenced to determine the rate of mutant codonincorporation. The number of fragments sequenced will be based on thenumber of target sites mutated and library validation will be determinedby the observed rate of mutation at targeted sites (Table 10). The rateof vectors without inserts should be less than 2%. The rate of mutationat non-targeted sites should be less than 8%.

The ability to generate the desired level of efficiency of mutagenesisby this approach will be determined by sequencing of a subset of clones.The alternative to BAG will be using a “DNA shuffle” protocol. Thisrequires pooling all of the mutants, single, double, triple, etc.Following DNA preparation, Fc regions will be amplified by PCR usingflanking primers that selectively amplify the mutated region of the Fc,˜700 bp. Novel mutants are constructed by reshuffling of mutations inthe Fc via DNAseI treatment of the amplified DNA and isolation of150-200 bp fragments (see, e.g., Stemmer et al., 1994, Proc. Natl. Acad.Sci. U.S.A. 91: 10747-51). Fragments will be religated, PCR amplifiedwith nested primers and cloned into the yeast surface display vector,pYD 1. The recombined library will be reselected in the yeast Fc displayscreen as described and exemplified herein.

BAG libraries will utilize most of the same equipment as thecombinatorial library. However cloning will be in a vector suitable foryeast surface display and will not require arraying of individual clonesas the yeast surface display will initially be employed for enrichmentof large libraries. Subsequent to the appropriate level of enrichmentindividual clones will be arrayed.

An initial library of molecules comprising variant Fc regions isproduced using any random based mutagenesis techniques known in the art.It will be appreciated by one of skill in the art that amino acidsequence variants of Fc regions may be obtained by any mutagenesistechnique known to those skilled in the art. Some of these techniquesare briefly described herein, however, it will be recognized thatalternative procedures may produce an equivalent result. In a preferredembodiment molecules of the invention comprising variant Fc regions areprepared by error-prone PCR as exemplified in Example 6, infra (SeeLeung et al., 1989, Technique, 1:11). It is especially preferred to haveerror rates of 2-3 bp/Kb for use in the methods of the invention. In oneembodiment, using error prone PCR a mutation frequency of 2-3mutations/kb is obtained.

Mutagenesis may be performed in accordance with any of the techniquesknown in the art including, but not limited to, synthesizing anoligonucleotide having one or more modifications within the sequence ofthe Fc region of an antibody or a polypeptide comprising an Fc region(e.g., the CH2 or CH3 domain) to be modified. Site-specific mutagenesisallows the production of mutants through the use of specificoligonucleotide sequences which encode the DNA sequence of the desiredmutation, as well as a sufficient number of adjacent nucleotides, toprovide a primer sequence of sufficient size and sequence complexity toform a stable duplex on both sides of the deletion junction beingtraversed. Typically, a primer of about 30 to about 45 nucleotides ormore in length is preferred, with about 10 to about 25 or more residueson both sides of the junction of the sequence being altered. A number ofsuch primers introducing a variety of different mutations at one or morepositions may be used to generated a library of mutants.

The technique of site-specific mutagenesis is well known in the art, asexemplified by various publications (see, e.g., Kunkel et al., MethodsEnzymol., 154:367-82, 1987, which is hereby incorporated by reference inits entirety). In general, site-directed mutagenesis is performed byfirst obtaining a single-stranded vector or melting apart of two strandsof a double stranded vector which includes within its sequence a DNAsequence which encodes the desired peptide. An oligonucleotide primerbearing the desired mutated sequence is prepared, generallysynthetically. This primer is then annealed with the single-strandedvector, and subjected to DNA polymerizing enzymes such as T7 DNApolymerase, in order to complete the synthesis of the mutation-bearingstrand. Thus, a heteroduplex is formed wherein one strand encodes theoriginal non-mutated sequence and the second strand bears the desiredmutation. This heteroduplex vector is then used to transform ortransfect appropriate cells, such as E. coli cells, and clones areselected which include recombinant vectors bearing the mutated sequencearrangement. As will be appreciated, the technique typically employs aphage vector which exists in both a single stranded and double strandedform. Typical vectors useful in site-directed mutagenesis includevectors such as the M13 phage. These phage are readily commerciallyavailable and their use is generally well known to those skilled in theart. Double stranded plasmids are also routinely employed in sitedirected mutagenesis which eliminates the step of transferring the geneof interest from a plasmid to a phage.

Alternatively, the use of PCR™ with commercially available thermostableenzymes such as Taq DNA polymerase may be used to incorporate amutagenic oligonucleotide primer into an amplified DNA fragment that canthen be cloned into an appropriate cloning or expression vector. See,e.g., Tomic et al., Nucleic Acids Res., 18(6):1656, 1987, and Upender etal., Biotechniques, 18(1):29-30, 32, 1995, for PCR™—mediated mutagenesisprocedures, which are hereby incorporated in their entireties. PCR™employing a thermostable ligase in addition to a thermostable polymerasemay also be used to incorporate a phosphorylated mutagenicoligonucleotide into an amplified DNA fragment that may then be clonedinto an appropriate cloning or expression vector (see e.g., Michael,Biotechniques, 16(3):410-2, 1994, which is hereby incorporated byreference in its entirety).

Another method for preparing variants for use in the invention, iscassette mutagenesis based on the technique described by Wells et al.(1985, Gene, 34: 315). The starting material is the plasmid comprisingthe desired DNA encoding the protein to be mutated (e.g., the DNAencoding a polypeptide comprising an Fc region). The codon(s) in the DNAsequence to be mutated are identified; there must be a uniquerestriction endonuclease site on each side of the identified mutationssite(s). If no such restriction site exits, it may be generated byoligonucleotide directed mutagenesis. After the restriction sites havebeen introduced into the plasmid, the plasmid is cut at these sites andlinearized. A double-stranded oligonucleotide encoding the sequence ofthe DNA between the restriction sites but containing the mutation issynthesized using standard procedures known to those skilled in the art.The double stranded oligonucleotide is referred to as the cassette. Thiscassette is designed to have 3′ and 5′ ends that are compatible with theends of the linearized plasmid, such that it can be directly ligated tothe plasmid.

Other methods known to those of skill in the art for producing sequencevariants of the Fc region of an antibody or polypeptides comprising anFc region can be used. For example, recombinant vectors encoding theamino acid sequence of the constant domain of an antibody or a fragmentthereof may be treated with mutagenic agents, such as hydroxylamine, toobtain sequence variants.

Once a mutant library is produced according to the methods described,the mutagenized library is transformed into a yeast strain, preferablyEBY100 (Invitrogen), MATα ura3-52 trpl leu2Δl his3Δ200 pep4::HIS3prb1Δ1.6R can1 GAL::GAL-AGA1 using a standard lithium acetatetransformation protocol known to those skilled in the art.

It will be appreciated by one of skill in the art, that once moleculesof the invention with desired effector function and/or bindingproperties (e.g., molecules with variant Fc regions with at least oneamino acid modification, which modification confers ADCC activity orenhances the affinity of the variant Fc region for FcγRIIIA relative toa comparable molecule, comprising a wild-type Fc region) have beenidentified (See Section 5.1 and Table 2) according to the methods of theinvention, other molecules (i.e, therapeutic antibodies) may beengineered using standard recombinant DNA techniques and any knownmutagenesis techniques, as described in this section to produceengineered molecules carrying the identified mutation sites.

5.2.4 Yeast Surface Display

The preferred method for screening and identifying molecules comprisingvariant Fc regions with altered FcγR affinities (i.e., enhanced FcγRIIIAaffinity and/or FcγRIIA) is yeast surface display technology (for reviewsee Boder and Wittrup, 2000, Methods in Enzymology, 328: 430-444, whichis incorporated herein by reference in its entirety) which addresses thedeficiency in the prior art for screening binding interactions ofextracellular post-translationally modified proteins. Specifically, theyeast surface display is a genetic method whereby polypeptidescomprising Fc mutants are expressed on the yeast cell wall in a formaccessible for interacting with FcγR. Yeast surface display of themutant Fc containing polypeptides of the invention may be performed inaccordance with any of the techniques known to those skilled in the art.See U.S. Pat. Nos. 6,423,538; 6,114,147; and 6,300,065, all of which areincorporated herein by reference in their entirety. See Boder et al.,1997 Nat. Biotechnol., 15:553-7; Boder et al., 1998 Biotechnol. Prog.,14:55-62; Boder et al., 2000 Methods Enzymol., 328:430-44; Boder et al.,2000 Proc. Natl. Acad. Sci. U.S.A., 2000, 97:10701-5; Shusta et al.,1998 Nat. Biotechnol., 1998, 16:773-7; Shusta et al., 1999 J. Mol.Biol., 292:949-56; Shusta et al., 1999 Curr. Opin. Biotechnol.,10:117-22; Shusta et al., 2000 Nat. Biotechnol., 18:754-9; Wittrup etal., 1994 Ann. N.Y. Acad. Sci., 745:321-30; Wittrup et al., 1994Cytometry, 16:206-13; Wittrup, 1995 Curr. Opin. Biotechnol., 6:203-8;Wittrup, 1999 Trends Biotechnol., 17:423-4; Wittrup, 2000 Nat.Biotechnol., 18:1039-40; Wittrup, 2001 Curr. Opin. Biotechnol.,12:395-9.

Yeast Surface Display will be used to enrich libraries containing >10⁷independent clones. This approach will provide the ability to enrichlarge libraries >20-fold in single sort. Fc mutant librarieswith >10,000 independent mutants (4 or more sites) will be cloned intothe appropriate vectors for yeast surface display and enriched by FACSsorting until <8000 mutants are able to be tested by other biochemicaland functional assays as described below.

The invention provides methods for constructing an Fc mutant library inyeast for displaying molecules comprising Fc regions, which have beenmutated as described in Section 5.2.2. Preferably, the Fc mutantlibraries for use in the methods of the invention contain at least 10⁷cells, up to 10⁹ cells. One exemplary method for constructing a Fclibrary for use in the methods of the invention comprises the following:nucleic acids encoding molecules comprising Fc regions are cloned intothe multiple cloning site of a vector derived from a yeast replicatingvector, e.g., pCT302; such that the Fc encoding nucleic acids areexpressed under the control of the GAL1 galactose-inducible promoter andin-frame with a nucleotide sequence encoding Aga2p, the matingagglutinin cell wall protein. In a preferred embodiment, nucleic acidsencoding molecules comprising Fc regions are cloned C-terminal to theAga2p coding region, such that a Fc-region Aga2p fusion protein isencoded. A fusion protein comprising the Aga2p protein and polypeptidescomprising Fc regions will be secreted extracellularly and displayed onthe cell wall via disulfide linkage to the Aga1p protein, an integralcell wall protein, using the preferred construct of the invention. In analternative embodiment, the constructs may further comprise nucleotidesequences encoding epitope tags. Any epitope tag nucleotide codingsequence known to those skilled in the art can be used in accordancewith the invention, including, but not limited to nucleotide sequencesencoding hemagglutinin (HA), c-myc Xpress TAG, His-TAG, or V5TAG. Thepresence of the fusion protein on the yeast cell surface may be detectedusing FACS analysis, confocal fluorescence microscopy or standardimmunostaining methods, all of which are known to those skilled in theart. In one embodiment, the presence of the Fc fusion proteins of theinvention on the yeast cell surface are detected using Fc-specificmonoclonal antibodies (CH3 specific), including but not limited to IgG1Fc-specific monoclonal antibody, HP6017 (Sigma), JL512 (Immunotech), andany antibody disclosed in Partridge et al., 1986, Molecular Immunology,23 (12): 1365-72, which is incorporated herein by reference in itsentirety. In another embodiment, the presence of the Fc fusion proteinsof the invention are detected by immunofluorescent labeling of epitopetags using techniques known to those skilled in the art. It isparticularly useful in the methods of the invention, to use nucleotidesequences encoding epitope tags to flank the nucleic acids encoding theFc fusion proteins, as an internal control, to detect if the fusionproteins are displayed on the cell wall in a partially proteolyzed form.

5.2.5 Screening of Yeast Display Libraries

The invention encompasses screening the yeast display libraries usingimmunological based assays including but not limited to cell basedassays, solution based assays, and solid phase based assays.

In some embodiments, the invention encompasses identification of Fcmutants with altered FcγR affinities using affinity maturation methodswhich are known to those skilled in the art and encompassed herein.Briefly, affinity maturation creates novel alleles by randomlyrecombining individual mutations present in a mutant library, see, e.g.,Hawkins et al., 1992, J. Mol. Biol. 226: 889-896; Stemmer et al., 1994Nature, 370: 389-91; both of which are incorporated herein by referencein their entireties. It has been used successfully to increase theaffinity of antibodies, T cell receptors and other proteins.

The invention encompasses using mutations that show increased FcγRbinding as a baseline to construct new mutant libraries with enhancedphenotypes. Using the methods of the invention, a population of IgG1 Fcmutants enriched by yeast surface display for increased binding to anFcγR, e.g., FcγRIIIA, may be selected. Following DNA preparation, Fcregions can be amplified by PCR using flanking primers that selectivelyamplify the mutated region of the Fc, which is about ˜700 bp usingmethods known to one skilled in the art and exemplified or disclosedherein. Novel mutants can thus be constructed by reshuffling ofmutations in the Fc region for example via DNAseI treatment of theamplified DNA and isolation of fragments using methods such as thosedisclosed by Stemmer et al., 1994 Proc. Natl. Acad. Sci. USA 91:10747-51, which is incorporated herein by reference in its entirety.Fragments can then be religated, PCR amplified with nested primers andcloned into the yeast display vector, e.g., pYD1 using methods known toone skilled in the art. The recombined library can then be reselected inthe yeast Fc display screen. As the K_(D) decreases, below 10 nM,conditions can be established to allow for further increases in affinitybased on the reduction of the off rate of the FcγRIIIA ligand from theFc receptor using methods known in the art such as those disclosed inBoder et al., 1998, Biotechnol. Prog. 14: 55-62, which is incorporatedherein by reference in its entirety. The invention encompasses a kineticscreen of the yeast library. A kinetic screen may be established bylabeling of the Fc displaying cells to saturation with a labeled ligand,e.g., a fluorescent ligand followed by incubation with an excess ofnon-labeled ligand for a predetermined period. After termination of thereaction by the addition of excess buffer (e.g., 1×PBS, 0.5 mg/ml BSA)cells will be analyzed by FACS and sort gates set for selection. Aftereach round of enrichment individual mutants can be tested for foldincreases in affinity and sequenced for diversity. The in vitrorecombination process can be repeated. In some embodiments, the in vitrorecombination process is repeated at least 3 times.

Selection of the Fc variants of the invention may be done using any FcγRincluding but not limited to polymorphic variants of FcγR. In someembodiments, selection of the Fc variants is done using a polymorphicvariant of FcγRIIIA which contains a phenylalanine at position 158. Inother embodiments, selection of the Fc variants is done using apolymorphic variant of FcγRIIIA which contains a valine at position 158.FcγRIIIA 158V displays a higher affinity for IgG1 than 158F and anincreased ADCC activity (see, e.g., Koene et al., 1997, Blood,90:1109-14; Wu et al., 1997, J. Clin. Invest. 100: 1059-70, both ofwhich are incorporated herein by reference in their entireties); thisresidue in fact directly interacts with the lower hinge region of IgG1as recently shown by IgG1-FcγRIIIA co-crystallization studies, see,e.g., Sonderman et al., 2000, Nature, 100: 1059-70, which isincorporated herein by reference in its entirety. Studies have shownthat in some cases therapeutic antibodies have improved efficacy inFcγRIIIA-158V homozygous patients. Therapeutic antibodies may also bemore effective in patients heterozygous for FcγRIIIA-158V andFcγRIIIA-158F, and in patients with FcγRIIA H131.

The invention encompasses screening yeast libraries based on FcγRIIBdepletion and FcγRIIIA selection so that Fc mutants are selected thatnot only have an enhanced affinity for FcγRIIIIA but also have a reducedaffinity for FcγRIIB. Yeast libraries may be enriched for clones thathave a reduced affinity for FcγRIIB by sequential depletion methods, forexample, by incubating the yeast library with magnetic beads coated withFcγRIIB. FcγRIIB depletion is preferably carried out sequentially sothat the library is enriched in clones that have a reduced affinity forFcγRIIB. In some embodiments, the FcγRIIB depletion step results in apopulation of cells so that only 30%, preferably only 10%, morepreferably only 5%, most preferably less than 1% bind FcγRIIB. In someembodiments, FcγRIIB depletion is carried out in at least 3 cycles, atleast 4 cycles, at least 6 cycles. The FcγRIIB depletion step ispreferably combined with an FcγRIIIIA selection step, for example usingFACS sorting so that Fc variants with an enhanced affinity for FcγRIIIIAare selected.

5.2.5.1 FACs Assays; Solid Phased Assays and Immunological Based Assays

The invention encompasses characterization of the mutant Fc fusionproteins that are displayed on the yeast surface cell wall, according tothe methods described in Section 5.2.3. One aspect of the inventionprovides a method for selecting mutant Fc fusion proteins with adesirable binding property, specifically, the ability of the mutant Fcfusion protein to bind FcγRIIIA and/or FcγRIIA with a greater affinitythan a comparable polypeptide comprising a wild-type Fc region bindsFcγRIIIA and/or FcγRIIA. In another embodiment, the invention provides amethod for selecting mutant Fc fusion proteins with a desirable bindingproperty, specifically, the ability of the mutant Fc fusion protein tobind FcγRIIIA and/or FcγRIIA with a greater affinity than a comparablepolypeptide comprising a wild-type Fc region binds FcγRIIIA and/orFcγRIIA, and further the ability of the mutant Fc fusion protein to bindFcγRIIB with a lower affinity than a comparable polypeptide comprising awild-type Fc region binds FcγRIIB. It will be appreciated by one skilledin the art, that the methods of the invention can be used foridentifying and screening any mutations in the Fc regions of molecules,with any desired binding characteristic.

Yeast cells displaying the mutant Fc fusion proteins can be screened andcharacterized by any biochemical or immunological based assays known tothose skilled in the art for assessing binding interactions.

Preferably, fluorescence activated cell sorting (FACS), using any of thetechniques known to those skilled in the art, is used for screening themutant Fc fusion proteins displayed on the yeast cell surface forbinding FcγRIIIA, preferably the FcγRIIIA tetrameric complex, oroptionally FcγRIIB. Flow sorters are capable of rapidly examining alarge number of individual cells that contain library inserts (e.g.,10-100 million cells per hour) (Shapiro et al., Practical FlowCytometry, 1995). Additionally, specific parameters used foroptimization including, but not limited to, ligand concentration (i.e.,FcγRIIIA tetrameric complex), kinetic competition time, or FACSstringency may be varied in order to select for the cells which displayFc fusion proteins with specific binding properties, e.g., higheraffinity for FcγRIIIA compared to a comparable polypeptide comprising awild-type Fc region. Flow cytometers for sorting and examiningbiological cells are well known in the art. Known flow cytometers aredescribed, for example, in U.S. Pat. Nos. 4,347,935; 5,464,581;5,483,469; 5,602,039; 5,643,796; and 6,211,477; the entire contents ofwhich are incorporated by reference herein. Other known flow cytometersare the FACS Vantage™ system manufactured by Becton Dickinson andCompany, and the COPAS™ system manufactured by Union Biometrica.

According to a preferred embodiment of the invention, yeast cells areanalyzed by fluorescence activated cell sorting (FACS). In mostpreferred embodiments, the FACS analysis of the yeast cells is done inan iterative manner, at least twice, at least three times, or at least 5times. Between each round of selection cells are regrown and induced sothe Fc regions are displayed on the maximum number of yeast cellsurfaces. Although not intending to be bound by a particular mode ofaction, this iterative process helps enrich the population of the cellswith a particular phenotype, e.g., high binding to FcγRIIIA.

In preferred embodiments, screening for Fc variants of the inventioncomprises a selection process that has multiple rounds of screening,e.g., at least two rounds of screening. In one embodiment, screening forFc variants that have an enhanced affinity for FcγRIIIA may comprise thefollowing steps: in the first round of screening, a library of yeastcells, e.g., a naïve library of 10⁷ cells is enriched by FACS,preferably in an iterative manner, using for example labeled tetramericFcγRIIIA to select for Fc variants that have an enhanced affinity forFcγRIIIA; the variant Fc region that is selected with the desiredphenotype, e.g., enhanced binding to FcγRIIIA, is then introduced intoan antibody, e.g., a 4-4-20 antibody, and the engineered antibody isassayed using a secondary screen, e.g., ELISA for binding to an FcγR. Inthe second round of screening, a single mutation library may begenerated based on the first screen so that the Fc region harbors thevariant displaying the enhanced affinity for FcγRIIIA; and enriched byFACS using for example labeled monomeric FcγRIIIA in both the presenceand absence of unlabeled receptor; and the variant Fc region is thenintroduced into an antibody, e.g., a 4-4-20 antibody, and the engineeredantibody is assayed using a secondary screen, e.g., ELISA for binding toan FcγR. In some embodiments, the secondary screen may further comprisecharacterizing the antibodies comprising Fc variants in an ADCC orBIAcore based assay using methods disclosed herein

The invention encompasses FACS screening of the mutant yeast libraryunder equilibrium or kinetic conditions. When the screening is performedunder equilibrium conditions, an excess of the yeast library carrying Fcmutants is incubated with FcγRIIIA, preferably labeled FcγRIIIA at aconcentration 5-10 fold below the Kd, for at least one hour to allowbinding of Fc mutants to FcγRIIIA under equilibrium conditions. When thescreening is performed under kinetic conditions, the mutant yeastlibrary is incubated with labeled FcγRIIIA; the cells are then incubatedwith equimolar unlabeled FcγRIIIA for a pre-selected time, boundFcγRIIIA is then monitored.

One exemplary method of analyzing the yeast cells expressing mutant Fcfusion proteins with FACS is costaining the cells withFcγRIIIA-tetrameric complex which has been labeled with a fluorescentlabel such as, PE and an anti-Fc antibody, such as F(ab)₂ anti-Fc whichhas been fluorescently labeled. Fluorescence measurements of a yeastlibrary produced according to the methods of the invention preferablyinvolves comparisons with controls; for example, yeast cells that lackthe insert encoding molecules comprising an Fc region (negativecontrol). The flow sorter has the ability not only to measurefluorescence signals in cells at a rapid rate, but also to collect cellsthat have specified fluorescent properties. This feature may be employedin a preferred embodiment of the invention to enrich the initial librarypopulation for cells expressing Fc fusion proteins with specific bindingcharacteristics, e.g., higher affinity for FcγRIIIA compared to acomparable polypeptide comprising a wild-type Fc region. In a preferredembodiment of the invention, yeast cells are analyzed by FACS and sortgates established to select for cells that show the highest affinity forFcγRIIIA relative to the amount of Fc expression on the yeast cellsurface. According to a preferred embodiment, four consecutive sorts areestablished, wherein the gates for each successive sort is 5.5%, 1%,0.2%, and 0.1%. It is preferred that the yeast display library formedaccording to the methods of the invention be over-sampled by at least10-fold to improve the probability of isolating rare clones (e.g.,analyze ˜10⁸ cells from a library of 10⁷ clones). Alternatively, 2-5sorts are established to select for cells of the desired phenotype. Sortgates can be established empirically by one skilled in the art.

In other preferred embodiments, mutant Fc fusion proteins displayed onthe yeast cell surface are screened using solid phase based assays, forexample assays using magnetic beads, e.g., supplied by Dynal, preferablyin a high through put manner for binding to an FcγR, e.g., FcγRIIIA. Inone embodiment, magnetic bead assays may be used to identify mutantswith enhanced affinity for FcγRIIIA and/or reduced affinity for FcγRIIB.An exemplary assay to identify mutants with enhanced affinity forFcγRIIIA and reduced affinity for FcγRIIB may comprise selecting mutantsby a sequential solid phase depletion using magnetic beads coated withFcγRIIB followed by selection with magnetic beads coated with FcγRIIIA.For example, one assay may comprise the following steps: incubating thelibrary of yeast cells generated in accordance with the methods of theinvention with magnetic beads coated with FcγRIIB; separating yeastcells bound to beads from the non bound fraction by placing the mixturein a magnetic field, removing the non-bound yeast cells and placing themin a fresh media; binding the yeast cells to beads coated with FcγRIIIA,separating yeast cells bound to beads from the non bound fraction byplacing the mixture in a magnetic field, removing the non-bound yeastcells; removing the bound cells by rigorous vortexing; growing therecovered cells in glucose containing media; re-inducing in selectivemedia containing galactose. The selection process is repeated at leastonce. Inserts containing the Fc domain are then amplified using commonmethodologies known in the art, e.g., PCR, and introduced into anantibody by methods already described for further characterization.

In an alternative embodiment, a non-yeast based system is used tocharacterize the binding properties of the molecules of the invention.One exemplary system for characterizing the molecules of the inventioncomprises a mammalian expression vector containing the heavy chain ofthe anti-fluorescein monoclonal antibody 4-4-20, into which the nucleicacids encoding the molecules of the invention with variant Fc regionsare cloned. The resulting recombinant clone is expressed in a mammalianhost cell line (i.e., human kidney cell line 293H), and the resultingrecombinant immunoglobulin is analyzed for binding to FcγR using anystandard assay known to those in the art, including but not limited toELISA and FACS.

Molecules of the present invention may be characterized in a variety ofways. In particular, molecules of the invention comprising modified Fcregions may be assayed for the ability to immunospecifically bind to aligand, e.g., FcγRIIIA tetrameric complex. Such an assay may beperformed in solution (e.g., Houghten, Bio/Techniques, 13:412-421,1992), on beads (Lam, Nature, 354:82-84, 1991, on chips (Fodor, Nature,364:555-556, 1993), on bacteria (U.S. Pat. No. 5,223,409), on spores(U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (Cullet al., Proc. Natl. Acad. Sci. USA, 89:1865-1869, 1992) or on phage(Scott and Smith, Science, 249:386-390, 1990; Devlin, Science,249:404-406, 1990; Cwirla et al., Proc. Natl. Acad. Sci. USA,87:6378-6382, 1990; and Felici, J. Mol. Biol., 222:301-310, 1991) (eachof these references is incorporated by reference herein in itsentirety). Molecules that have been identified to immunospecificallybind to an ligand, e.g., FcγRIIIA can then be assayed for theirspecificity and affinity for the ligand.

Molecules of the invention that have been engineered to comprisemodified Fc regions (e.g., therapeutic antibodies) or have beenidentified in the yeast display system to have the desired phenotype(see Section 5.1) may be assayed for immunospecific binding to anantigen (e.g., cancer antigen and cross-reactivity with other antigens(e.g., FcγR) by any method known in the art. Immunoassays which can beused to analyze immunospecific binding and cross-reactivity include, butare not limited to, competitive and non-competitive assay systems usingtechniques such as western blots, radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al., eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).

The binding affinity of the molecules of the present inventioncomprising modified Fc regions to a ligand, e.g., FcγR tetramericcomplex and the off-rate of the interaction can be determined bycompetitive binding assays. One example of a competitive binding assayis a radioimmunoassay comprising the incubation of labeled ligand, suchas tetrameric FcγR (e.g., ³H or ¹²⁵I) with a molecule of interest (e.g.,molecules of the present invention comprising modified Fc regions) inthe presence of increasing amounts of unlabeled ligand, such astetrameric FcγR, and the detection of the molecule bound to the labeledligand. The affinity of the molecule of the present invention for theligand and the binding off-rates can be determined from the saturationdata by scatchard analysis.

In a preferred embodiment, BIAcore kinetic analysis is used to determinethe binding on and off rates of molecules of the present invention to aligand such as FcγR. BIAcore kinetic analysis comprises analyzing thebinding and dissociation of a ligand from chips with immobilizedmolecules (e.g., molecules comprising modified Fc regions) on theirsurface.

5.2.6 Sequencing of Mutants

Any of a variety of sequencing reactions known in the art can be used todirectly sequence the molecules of the invention comprising variant Fcregions. Examples of sequencing reactions include those based ontechniques developed by Maxim and Gilbert (Proc. Natl. Acad. Sci. USA,74:560, 1977) or Sanger (Proc. Natl. Acad. Sci. USA, 74:5463, 1977). Itis also contemplated that any of a variety of automated sequencingprocedures can be utilized (Bio/Techniques, 19:448, 1995), includingsequencing by mass spectrometry (see, e.g., PCT Publication No. WO94/16101, Cohen et al., Adv. Chromatogr., 36:127-162, 1996, and Griffinet al., Appl. Biochem. Biotechnol., 38:147-159, 1993).

5.2.7 Functional Assays of Molecules with Variant Fc Regions

The invention encompasses characterization of the molecules of theinvention (e.g., an antibody comprising a variant Fc region identifiedby the yeast display technology described supra; or therapeuticmonoclonal antibodies engineered according to the methods of theinvention) using assays known to those skilled in the art foridentifying the effector cell function of the molecules. In particular,the invention encompasses characterizing the molecules of the inventionfor FcγR-mediated effector cell function. Examples of effector cellfunctions that can be assayed in accordance with the invention, includebut are not limited to, antibody-dependent cell mediated cytotoxicity,phagocytosis, opsonization, opsonophagocytosis, C1q binding, andcomplement dependent cell mediated cytotoxicity. Any cell-based or cellfree assay known to those skilled in the art for determining effectorcell function activity can be used (For effector cell assays, seePerussia et al., 2000, Methods Mol. Biol. 121: 179-92; Baggiolini etal., 1998 Experientia, 44(10): 841-8; Lehmann et al., 2000 J. Immunol.Methods, 243(1-2): 229-42; Brown E J. 1994, Methods Cell Biol., 45:147-64; Munn et al., 1990 J. Exp. Med., 172: 231-237, Abdul-Majid etal., 2002 Scand. J. Immunol. 55: 70-81; Ding et al., 1998, Immunity8:403-411, each of which is incorporated by reference herein in itsentirety).

In one embodiment, the molecules of the invention can be assayed forFcγR-mediated phagocytosis in human monocytes. Alternatively, theFcγR-mediated phagocytosis of the molecules of the invention may beassayed in other phagocytes, e.g., neutrophils (polymorphonuclearleuckocytes; PMN); human peripheral blood monocytes, monocyte-derivedmacrophages, which can be obtained using standard procedures known tothose skilled in the art (e.g., see Brown E J. 1994, Methods Cell Biol.,45: 147-164). In one embodiment, the function of the molecules of theinvention is characterized by measuring the ability of THP-1 cells tophagocytose fluoresceinated IgG-opsonized sheep red blood cells (SRBC)by methods previously described (Tridandapani et al., 2000, J. Biol.Chem. 275:20480-7). For example, an exemplary assay for measuringphagocytosis of the molecules of the invention comprising variant Fcregions with enhanced affinities for FcγRIIIA, comprises of: treatingTHP-1 cells with a molecule of the invention or with a control antibodythat does not bind to FcγRIIIA, comparing the activity levels of saidcells, wherein a difference in the activities of the cells (e.g.,rosetting activity (the number of THP-1 cells binding IgG-coated SRBC),adherence activity (the total number of SRBC bound to THP-1 cells), andphagocytic rate) would indicate the functionality of the molecule of theinvention. It can be appreciated by one skilled in the art that thisexemplary assay can be used to assay any of the molecules identified bythe methods of the invention.

Another exemplary assay for determining the phagocytosis of themolecules of the invention is an antibody-dependent opsonophagocytosisassay (ADCP) which can comprise the following: coating a targetbioparticle such as Escherichia coli-labeled FITC (Molecular Probes) orStaphylococcus aureus-FITC with (i) wild-type 4-4-20 antibody, anantibody to fluorescein (See Bedzyk et al., 1989, J. Biol. Chem.,264(3): 1565-1569, which is incorporated herein by reference in itsentirety), as the control antibody for FcγR-dependent ADCP; or (ii)4-4-20 antibody harboring the D265A mutation that knocks out binding toFcγRIII, as a background control for FcγR-dependent ADCP (iii) 4-4-20antibody carrying variant Fc regions identified by the methods of theinvention and produced as exemplified in Example 6.6; and forming theopsonized particle; adding any of the opsonized particles described(i-iii) to THP-1 effector cells (a monocytic cell line available fromATCC) at a 1:1, 10:1, 30:1, 60:1, 75:1 or 100:1 ratio to allowFcγR-mediated phagocytosis to occur; preferably incubating the cells andE. coli-FITC/antibody at 37° C. for 1.5 hour; adding trypan blue afterincubation (preferably at room temperature for 2-3 min.) to the cells toquench the fluoroscence of the bacteria that are adhered to the outsideof the cell surface without being internalized; transferring cells intoa FACS buffer (e.g., 0.1%, BSA in PBS, 0.1%, sodium azide), analyzingthe fluorescence of the THP1 cells using FACS (e.g., BD FACS Calibur).Preferably, the THP-1 cells used in the assay are analyzed by FACS forexpression of FcγR on the cell surface. THP-1 cells express both CD32Aand CD64. CD64 is a high affinity FcγR that is blocked in conducting theADCP assay in accordance with the methods of the invention. The THP-1cells are preferably blocked with 100 μg/mL soluble IgG1 or 10% humanserum. To analyze the extent of ADCP, the gate is preferably set onTHP-1 cells and median fluorescence intensity is measured. The ADCPactivity for individual mutants is calculated and reported as anormalized value to the wild type chMab 4-4-20 obtained. The opsonizedparticles are added to THP-1 cells such that the ratio of the opsonizedparticles to THP-1 cells is 30:1 or 60:1. In most preferred embodiments,the ADCP assay is conducted with controls, such as E. coli-FITC inmedium, E. coli-FITC and THP-1 cells (to serve as FcγR-independent ADCPactivity), E. coli-FITC, THP-1 cells and wild-type 4-4-20 antibody (toserve as FcγR-dependent ADCP activity), E coli-FITC, THP-1 cells, 4-4-20D265A (to serve as the background control for FcγR-dependent ADCPactivity).

In another embodiment, the molecules of the invention can be assayed forFcγR-mediated ADCC activity in effector cells, e.g., natural killercells, using any of the standard methods known to those skilled in theart (See e.g., Perussia et al., 2000, Methods Mol. Biol. 121: 179-92;Weng et al., 2003, J. Clin. Oncol. 21:3940-3947). An exemplary assay fordetermining ADCC activity of the molecules of the invention is based ona ⁵¹Cr release assay comprising of: labeling target cells with[⁵¹Cr]Na₂CrO₄ (this cell-membrane permeable molecule is commonly usedfor labeling since it binds cytoplasmic proteins and althoughspontaneously released from the cells with slow kinetics, it is releasedmassively following target cell necrosis); opsonizing the target cellswith the molecules of the invention comprising variant Fc regions;combining the opsonized radiolabeled target cells with effector cells ina microtitre plate at an appropriate ratio of target cells to effectorcells; incubating the mixture of cells for 16-18 hours at 37° C.;collecting supernatants; and analyzing radioactivity. The cytotoxicityof the molecules of the invention can then be determined, for exampleusing the following formula: % lysis=(experimental cpm−target leakcpm)/(detergent lysis cpm−target leak cpm)×100%. Alternatively, %lysis=(ADCC-AICC)/(maximum release-spontaneous release). Specific lysiscan be calculated using the formula: specific lysis=% lysis with themolecules of the invention—% lysis in the absence of the molecules ofthe invention. A graph can be generated by varying either thetarget:effector cell ratio or antibody concentration.

In yet another embodiment, the molecules of the invention arecharacterized for antibody dependent cellular cytotoxicity (ADCC) see,e.g., Ding et al., Immunity, 1998, 8:403-11; which is incorporatedherein by reference in its entirety.

Preferably, the effector cells used in the ADCC assays of the inventionare peripheral blood mononuclear cells (PBMC) that are preferablypurified from normal human blood, using standard methods known to oneskilled in the art, e.g., using Ficoll-Paque density gradientcentrifugation. Preferred effector cells for use in the methods of theinvention express different FcγR activating receptors. The inventionencompasses, effector cells, THP-1, expressing FcγRI, FcγRIIA andFcγRIIB, and monocyte derived primary macrophages derived from wholehuman blood expressing both FcγRIIIA and FcγRIIB, to determine if Fcantibody mutants show increased ADCC activity and phagocytosis relativeto wild type IgG1 antibodies.

The human monocyte cell line, THP-1, activates phagocytosis throughexpression of the high affinity receptor FcγRI and the low affinityreceptor FcγRIIA (Fleit et al., 1991, J. Leuk. Biol. 49: 556). THP-1cells do not constitutively express FcγRIIA or FcγRIIB. Stimulation ofthese cells with cytokines effects the FcR expression pattern (Pricop etal., 2000 J. Immunol. 166: 531-7). Growth of THP-1 cells in the presenceof the cytokine IL4 induces FcγRIIB expression and causes a reduction inFcγRIIA and FcγRI expression. FcγRIIB expression can also be enhanced byincreased cell density (Tridandapani et al., 2002, J. Biol. Chem. 277:5082-9). In contrast, it has been reported that IFNγ can lead toexpression of FcγRIIIA (Pearse et al., 1993 PNAS USA 90: 4314-8). Thepresence or absence of receptors on the cell surface can be determinedby FACS using common methods known to one skilled in the art. Cytokineinduced expression of FcγR on the cell surface provides a system to testboth activation and inhibition in the presence of FcγRIIB. If THP-1cells are unable to express the FcγRIIB the invention also encompassesanother human monocyte cell line, U937. These cells have been shown toterminally differentiate into macrophages in the presence of IFNγ andTNF (Koren et al., 1979, Nature 279: 328-331).

FcγR dependent tumor cell killing is mediated by macrophage and NK cellsin mouse tumor models (Clynes et al., 1998, PNAS USA 95: 652-656). Theinvention encompasses the use of elutriated monocytes from donors aseffector cells to analyze the efficiency Fc mutants to trigger cellcytotoxicity of target cells in both phagocytosis and ADCC assays.Expression patterns of FcγRI, FcγRIIIA, and FcγRIIB are affected bydifferent growth conditions. FcγR expression from frozen elutriatedmonocytes, fresh elutriated monocytes, monocytes maintained in 10% FBS,and monocytes cultured in FBS+GM-CSF and or in human serum may bedetermined using common methods known to those skilled in the art. Forexample, cells can be stained with FcγR specific antibodies and analyzedby FACS to determine FcR profiles. Conditions that best mimic macrophagein vivo FcγR expression is then used for the methods of the invention.

In some embodiments, the invention encompasses the use of mouse cellsespecially when human cells with the right FcγR profiles are unable tobe obtained. In some embodiments, the invention encompasses the mousemacrophage cell line RAW264.7(ATCC) which can be transfected with humanFcγRIIIA and stable transfectants isolated using methods known in theart, see, e.g., Ralph et al., J. Immunol. 119: 950-4). Transfectants canbe quantitated for FcγRIIIA expression by FACS analysis using routineexperimentation and high expressors can be used in the ADCC assays ofthe invention. In other embodiments, the invention encompasses isolationof spleen peritoneal macrophage expressing human FcγR from knockouttransgenic mice such as those disclosed herein.

Lymphocytes may be harvested from peripheral blood of donors (PBM) usinga Ficoll-Paque gradient (Pharmacia). Within the isolated mononuclearpopulation of cells the majority of the ADCC activity occurs via thenatural killer cells (NK) containing FcγRIIIA but not FcγRIIB on theirsurface. Results with these cells indicate the efficacy of the mutantson triggering NK cell ADCC and establish the reagents to test withelutriated monocytes.

Target cells used in the ADCC assays of the invention include, but arenot limited to, breast cancer cell lines, e.g., SK-BR-3 with ATCCaccession number HTB-30 (see, e.g., Tremp et al., 1976, Cancer Res.33-41); B-lymphocytes; cells derived from Burkitts lymphoma, e.g., Rajicells with ATCC accession number CCL-86 (see, e.g., Epstein et al.,1965, J. Natl. Cancer Inst. 34: 231-240), and Daudi cells with ATCCaccession number CCL-213 (see, e.g., Klein et al., 1968, Cancer Res. 28:1300-10). The target cells must be recognized by the antigen bindingsite of the immunoglobulin to be assayed.

The ADCC assay is based on the ability of NK cells to mediate cell deathvia an apoptotic pathway. NK cells mediate cell death in part byFcγRIIIA's recognition of IgG bound to an antigen on a cell surface. TheADCC assays used in accordance with the methods of the invention may beradioactive based assays or fluorescence based assays. The ADCC assayused to characterize the molecules of the invention comprising variantFc regions comprises labeling target cells, e.g., SK-BR-3, MCF-7,OVCAR3, Raji, Daudi cells, opsonizing target cells with an antibody thatrecognizes a cell surface receptor on the target cell via its antigenbinding site; combining the labeled opsonized target cells and theeffector cells at an appropriate ratio, which can be determined byroutine experimentation; harvesting the cells; detecting the label inthe supernatant of the lysed target cells, using an appropriatedetection scheme based on the label used. The target cells may belabeled either with a radioactive label or a fluorescent label, usingstandard methods known in the art. For example the labels include, butare not limited to, [⁵¹Cr]Na₂CrO₄; and the acetoxymethyl ester of thefluorescence enhancing ligand, 2,2′:6′,2″-terpyridine-6-6″-dicarboxylate(TDA).

In a specific preferred embodiment, a time resolved fluorimetric assayis used for measuring ADCC activity against target cells that have beenlabeled with the acetoxymethyl ester of the fluorescence enhancingligand, 2,2′:6′,2″-terpyridine-6-6″-dicarboxylate (TDA). Suchfluorimetric assays are known in the art, e.g., see, Blomberg et al.,1996, Journal of Immunological Methods, 193: 199-206; which isincorporated herein by reference in its entirety. Briefly, target cellsare labeled with the membrane permeable acetoxymethyl diester of TDA(bis(acetoxymethyl) 2,2′:6′,2″-terpyridine-6-6″-dicarboxylate, (BATDA),which rapidly diffuses across the cell membrane of viable cells.Intracellular esterases split off the ester groups and the regeneratedmembrane impermeable TDA molecule is trapped inside the cell. Afterincubation of effector and target cells, e.g., for at least two hours,up to 3.5 hours, at 37° C., under 5% CO₂, the TDA released from thelysed target cells is chelated with Eu3+ and the fluorescence of theEuropium-TDA chelates formed is quantitated in a time-resolvedfluorometer (e.g., Victor 1420, Perkin Elmer/Wallac).

In another specific embodiment, the ADCC assay used to characterize themolecules of the invention comprising variant Fc regions comprises thefollowing steps: Preferably 4−5×10⁶ target cells (e.g., SK-BR-3, MCF-7,OVCAR3, Raji cells) are labeled with bis(acetoxymethyl)2,2′:6′,2″-terpyridine-t-6″-dicarboxylate (DELFIA BATDA Reagent, PerkinElmer/Wallac). For optimal labeling efficiency, the number of targetcells used in the ADCC assay should preferably not exceed 5×10⁶. BATDAreagent is added to the cells and the mixture is incubated at 37° C.preferably under 5% CO₂, for at least 30 minutes. The cells are thenwashed with a physiological buffer, e.g., PBS with 0.125 mMsulfinpyrazole, and media containing 0.125 mM sulfinpyrazole. Thelabeled target cells are then opsonized (coated) with a molecule of theinvention comprising a variant Fc region, i.e., an immunoglobulincomprising a variant Fc region of the invention, including, but notlimited to, a polyclonal antibody, a monoclonal antibody, a bispecificantibody, a multi-specific antibody, a humanized antibody, or a chimericantibody. In preferred embodiments, the immunoglobulin comprising avariant Fc region used in the ADCC assay is specific for a cell surfacereceptor, a tumor antigen, or a cancer antigen. The immunoglobulin intowhich a variant Fc region of the invention is introduced mayspecifically bind any cancer or tumor antigen, such as those listed insection 5.4. Additionally, the immunoglobulin into which a variant Fcregion of the invention is introduced may be any therapeutic antibodyspecific for a cancer antigen, such as those listed in section 5.4. Insome embodiments, the immunoglobulin comprising a variant Fc region usedin the ADCC assay is an anti-fluoresceine monoclonal antibody, 4-4-20(Kranz et al., 1982 J. Biol. Chem. 257(12): 6987-6995) a mouse-humanchimeric anti-CD20 monoclonal antibody 2H7 (Liu et al., 1987, Journal ofImmunology, 139: 3521-6); or a humanized antibody (Ab4D5) against thehuman epidermal growth factor receptor 2 (p185 HER2) (Carter et al.(1992, Proc. Natl. Acad. Sci. USA 89: 4285-9). The target cells in theADCC assay are chosen according to the immunoglobulin into which avariant Fc region of the invention has been introduced so that theimmunoglobulin binds a cell surface receptor of the target cellspecifically. Preferably, the ADCC assays of the invention are performedusing more than one engineered antibody, e.g., anti Her2/neu, 4-4-20,2B6, RITUXAN™, and 2H7, harboring the Fc variants of the invention. In amost preferred embodiment, the Fc variants of the invention areintroduced into at least 3 antibodies and their ADCC activities aretested. Although not intending to be bound by a particular mechanism ofaction, examining at least 3 antibodies in these functional assays willdiminish the chance of eliminating a viable Fc mutation erroneously.

Target cells are added to effector cells, e.g., PBMC, to produceeffector:target ratios of approximately 1:1, 10:1, 30:1, 50:1, 75:1, or100:1. In a specific embodiment, when the immunoglobulin comprising avariant Fc region has the variable domain of 4-4-20, the effector:targetis 75:1. The effector and target cells are incubated for at least twohours, up to 3.5 hours, at 37° C., under 5% CO₂. Cell supernatants areharvested and added to an acidic europium solution (e.g., DELFIAEuropium Solution, Perkin Elmer/Wallac). The fluorescence of theEuropium-TDA chelates formed is quantitated in a time-resolvedfluorometer (e.g., Victor 1420, Perkin Elmer/Wallac). Maximal release(MR) and spontaneous release (SR) are determined by incubation of targetcells with 1% TX-100 and media alone, respectively. Antibody independentcellular cytotoxicity (AICC) is measured by incubation of target andeffector cells in the absence of antibody. Each assay is preferablyperformed in triplicate. The mean percentage specific lysis iscalculated as: Experimental release (ADCC)-AICC)/(MR−SR)×100.

The invention encompasses characterization of the Fc variants in bothNK-dependent and macrophage dependent ADCC assays. Fc variants of theinvention have altered phenotypes such as an altered effector functionas assayed in an NK dependent or macrophage dependent assay.

The invention encompasses assays known in the art and exemplifiedherein, to bind C1q and mediate complement dependent cytotoxicity (CDC).To determine C1q binding, a C1q binding ELISA may be performed. Anexemplary assay may comprise the following: assay plates may be coatedovernight at 4° C. with polypeptide variant or starting polypeptide(control) in coating buffer. The plates may then be washed and blocked.Following washing, an aliquot of human C1q may be added to each well andincubated for 2 hrs at room temperature. Following a further wash, 100uL of a sheep anti-complement C1q peroxidase conjugated antibody may beadded to each well and incubated for 1 hour at room temperature. Theplate may again be washed with wash buffer and 100 ul of substratebuffer containing OPD (O-phenylenediamine dihydrochloride (Sigma)) maybe added to each well. The oxidation reaction, observed by theappearance of a yellow color, may be allowed to proceed for 30 minutesand stopped by the addition of 100 ul of 4.5 NH₂SO₄. The absorbance maythen read at (492-405) nm.

A preferred variant in accordance with the invention is one thatdisplays a significant reduction in C1q binding, as detected andmeasured in this assay or a similar assay. Preferably the moleculecomprising an Fc variant displays about 50 fold reduction, about 60fold, about 80 fold, or about 90 fold reduction in C1q binding comparedto a control antibody having a nonmutated IgG1 Fc region. In the mostpreferred embodiment, the molecule comprising an Fc variant does notbind C1q, i.e. the variant displays about 100 fold or more reduction inC1q binding compared to the control antibody.

Another exemplary variant is one which has a better binding affinity forhuman C1q than the molecule comprising wild type Fc region. Such amolecule may display, for example, about two-fold or more, andpreferably about five-fold or more, improvement in human C1q bindingcompared to the parent molecule comprising wild type Fc region. Forexample, human C1q binding may be about two-fold to about 500-fold, andpreferably from about two-fold or from about five-fold to about1000-fold improved compared to the molecule comprising wild type Fcregion.

To assess complement activation, a complement dependent cytotoxicity(CDC) assay may be performed, e.g. as described in Gazzano-Santoro etal., J. Immunol. Methods 202:163 (1996), which is incorporated herein byreference in its entirety. Briefly, various concentrations of themolecule comprising a variant Fc region and human complement may bediluted with buffer. Cells which express the antigen to which themolecule comprising a variant Fc region binds may be diluted to adensity of about 1×10⁶ cells/ml. Mixtures of the molecule comprising avariant Fc region, diluted human complement and cells expressing theantigen may be added to a flat bottom tissue culture 96 well plate andallowed to incubate for 2 hrs at 37° C. and 5% CO₂ to facilitatecomplement mediated cell lysis. 50 uL of alamar blue (AccumedInternational) may then be added to each well and incubated overnight at37° C. The absorbance is measured using a 96-well fluorometer withexcitation at 530 nm and emission at 590 nm. The results may beexpressed in relative fluorescence units (RFU). The sampleconcentrations may be computed from a standard curve and the percentactivity as compared to nonvariant molecule, i.e., a molecule comprisingwild type Fc region, is reported for the variant of interest.

In some embodiments, an Fc variant of the invention does not activatecomplement. Preferably the variant does not appear to have any CDCactivity in the above CDC assay. The invention also pertains to avariant with enhanced CDC compared to a parent molecule (a moleculecomprising wild type Fc region), e.g., displaying about two-fold toabout 100-fold improvement in CDC activity in vitro or in vivo (e.g., atthe IC50 values for each molecule being compared). Complement assays maybe performed with guinea pig, rabbit or human serum. Complement lysis oftarget cells may be detected by monitoring the release of intracellularenzymes such as lactate dehydrogenase (LDH), as described inKorzeniewski et al., 1983 Immunol. Methods 64(3): 313-20; and Decker etal., 1988, J. Immunol. Methods 115(1): 61-9, each of which isincorporated herein by reference in its entirety; or the release of anintracellular lable such as europium, chromium 51 or indium 111 in whichtarget cells are labeled as described herein.

5.2.8 Other Assays

The molecules of the invention comprising variant Fc regions may also beassayed using any surface plasmon resonance based assays known in theart for characterizing the kinetic parameters of Fc-FcγR interactionbinding. Any SPR instrument commercially available including, but notlimited to, BIAcore Instruments, available from Biacore AB (Uppsala,Sweden); IAsys instruments available from Affinity Sensors (Franklin,Mass.); IBIS system available from Windsor Scientific Limited (Berks,UK), SPR-CELLIA systems available from Nippon Laser and Electronics Lab(Hokkaido, Japan), and SPR Detector Spreeta available from TexasInstruments (Dallas, Tex.) can be used in the instant invention. For areview of SPR-based technology see Mullet et al., 2000, Methods 22:77-91; Dong et al., 2002, Review in Mol. Biotech., 82: 303-23; Fivash etal., 1998, Current Opinion in Biotechnology 9: 97-101; Rich et al.,2000, Current Opinion in Biotechnology 11: 54-61; all of which areincorporated herein by reference in their entirety. Additionally, any ofthe SPR instruments and SPR based methods for measuring protein-proteininteractions described in U.S. Pat. Nos. 6,373,577; 6,289,286;5,322,798; 5,341,215; and 6,268,125 are contemplated in the methods ofthe invention, all of which are incorporated herein by reference intheir entirety.

Briefly, SPR based assays involve immobilizing a member of a bindingpair on a surface, and monitoring its interaction with the other memberof the binding pair in solution in real time. SPR is based on measuringthe change in refractive index of the solvent near the surface thatoccurs upon complex formation or dissociation. The surface onto whichthe immobilization occur is the sensor chip, which is at the heart ofthe SPR technology; it consists of a glass surface coated with a thinlayer of gold and forms the basis for a range of specialized surfacesdesigned to optimize the binding of a molecule to the surface. A varietyof sensor chips are commercially available especially from the companieslisted supra, all of which may be used in the methods of the invention.Examples of sensor chips include those available from BIAcore AB, Inc.,e.g., Sensor Chip CMS, SA, NTA, and HPA. A molecule of the invention maybe immobilized onto the surface of a sensor chip using any of theimmobilization methods and chemistries known in the art, including butnot limited to, direct covalent coupling via amine groups, directcovalent coupling via sulfhydryl groups, biotin attachment to avidincoated surface, aldehyde coupling to carbohydrate groups, and attachmentthrough the histidine tag with NTA chips.

In some embodiments, the kinetic parameters of the binding of a moleculeof the invention comprising variant Fc regions, e.g., immunoglobulinscomprising variant Fc region, to an FcγR may be determined using aBIAcore instrument (e.g., BIAcore instrument 1000, BIAcore Inc.,Piscataway, N.J.). Any FcγR can be used to assess the interaction withthe molecules of the invention comprising variant Fc regions. In aspecific embodiment the FcγR is FcγRIIIA, preferably a soluble monomericFcγRIIIA. For example, in one embodiment, the soluble monomeric FcγRIIIAis the extracellular region of FcγRIIIA joined to the linker-AVITAGsequence (see, U.S. Provisional Application No. 60/439,498, filed onJan. 9, 2003 (Attorney Docket No. 11183-004-888) and U.S. ProvisionalApplication No. 60/456,041 filed on Mar. 19, 2003, which areincorporated herein by reference in their entireties). In anotherspecific embodiment, the FcγR is FcγRIIB, preferably a soluble dimericFcγRIIB. For example, in one embodiment, the soluble dimeric FcγRIIBprotein is prepared in accordance with the methodology described in U.S.Provisional application No. 60/439,709 filed on Jan. 13, 2003, which isincorporated herein by reference in its entirety.

An exemplary assay for determining the kinetic parameters of a moleculecomprising a variant Fc region, wherein the molecule is the 4-4-20antibody, to an FcγR using a BIAcore instrument comprises the following:BSA-FITC is immobilized on one of the four flow cells of a sensor chipsurface, preferably through amine coupling chemistry such that about5000 response units (RU) of BSA-FITC is immobilized on the surface. Oncea suitable surface is prepared, 4-4-20 antibodies carrying the Fcmutations are passed over the surface, preferably by one minuteinjections of a 20 μg/mL solution at a 5 μL/mL flow rate. The level of4-4-20 antibodies bound to the surface ranges between 400 and 700 RU.Next, dilution series of the receptor (FcγRIIA and FcγRIIB-Fc fusionprotein) in HBS-P buffer (20 mM HEPES, 150 mM NaCl, 3 mM EDTA, pH 7.5)are injected onto the surface at 100 μL/min Antibody regenerationbetween different receptor dilutions is carried out preferably by single5 second injections of 100 mM NaHCO₃ pH 9.4; 3M NaCl. Any regenerationtechnique known in the art is contemplated in the method of theinvention.

Once an entire data set is collected, the resulting binding curves areglobally fitted using computer algorithms supplied by the SPR instrumentmanufacturer, e.g., BIAcore, Inc. (Piscataway, N.J.). These algorithmscalculate both the K_(on) and K_(off), from which the apparentequilibrium binding constant, K_(d) is deduced as the ratio of the tworate constants (i.e., K_(off)/K_(on)). More detailed treatments of howthe individual rate constants are derived can be found in theBlAevaluaion Software Handbook (BIAcore, Inc., Piscataway, N.J.). Theanalysis of the generated data may be done using any method known in theart. For a review of the various methods of interpretation of thekinetic data generated see Myszka, 1997, Current Opinion inBiotechnology 8: 50-7; Fisher et al., 1994, Current Opinion inBiotechnology 5: 389-95; O'Shannessy, 1994, Current Opinion inBiotechnology, 5:65-71; Chaiken et al., 1992, Analytical Biochemistry,201: 197-210; Morton et al., 1995, Analytical Biochemistry 227: 176-85;O'Shannessy et al., 1996, Analytical Biochemistry 236: 275-83; all ofwhich are incorporated herein by reference in their entirety.

In preferred embodiments, the kinetic parameters determined using an SPRanalysis, e.g., BIAcore, may be used as a predictive measure of how amolecule of the invention will function in a functional assay, e.g.,ADCC. An exemplary method for predicting the efficacy of a molecule ofthe invention based on kinetic parameters obtained from an SPR analysismay comprise the following: determining the K_(off) values for bindingof a molecule of the invention to FcγRIIIA and FcγRIIB; plotting (1)K_(off) (wt)/K_(off) (mut) for FcγRIIIA; (2) K_(off) (mut)/K_(off) (wt)for FcγRIIB against the ADCC data. Numbers higher than one show adecreased dissociation rate for FcγRIIIA and an increased dissociationrate for FcγRIIB relative to wild type; and possess and enhanced ADCCfunction.

5.3 Methods of Recombinantly Producing Molecules of the Invention

5.3.1 Polynucleotides Encoding Molecules of the Invention

The present invention also includes polynucleotides that encode themolecules, including the polypeptides and antibodies, of the inventionidentified by the methods of the invention. The polynucleotides encodingthe molecules of the invention may be obtained, and the nucleotidesequence of the polynucleotides determined, by any method known in theart.

Once the nucleotide sequence of the molecules (e.g., antibodies) thatare identified by the methods of the invention is determined, thenucleotide sequence may be manipulated using methods well known in theart, e.g., recombinant DNA techniques, site directed mutagenesis, PCR,etc. (see, for example, the techniques described in Sambrook et al.,2001, Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.; and Ausubel et al., eds.,1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY,which are both incorporated by reference herein in their entireties), togenerate, for example, antibodies having a different amino acidsequence, for example by generating amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, when the nucleic acids encode antibodies, oneor more of the CDRs are inserted within framework regions using routinerecombinant DNA techniques. The framework regions may be naturallyoccurring or consensus framework regions, and preferably human frameworkregions (see, e.g., Chothia et al., 1998, J. Mol. Biol. 278: 457-479 fora listing of human framework regions).

In another embodiment, human libraries or any other libraries availablein the art, can be screened by standard techniques known in the art, toclone the nucleic acids encoding the molecules of the invention.

5.3.2 Recombinant Expression of Molecules of the Invention

Once a nucleic acid sequence encoding molecules of the invention (i.e.,antibodies) has been obtained, the vector for the production of themolecules may be produced by recombinant DNA technology using techniqueswell known in the art. Methods which are well known to those skilled inthe art can be used to construct expression vectors containing thecoding sequences for the molecules of the invention and appropriatetranscriptional and translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, for example, thetechniques described in Sambrook et al., 1990, Molecular Cloning, ALaboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. and Ausubel et al. eds., 1998, Current Protocols inMolecular Biology, John Wiley & Sons, NY).

An expression vector comprising the nucleotide sequence of a moleculeidentified by the methods of the invention (i.e., an antibody) can betransferred to a host cell by conventional techniques (e.g.,electroporation, liposomal transfection, and calcium phosphateprecipitation) and the transfected cells are then cultured byconventional techniques to produce the molecules of the invention. Inspecific embodiments, the expression of the molecules of the inventionis regulated by a constitutive, an inducible or a tissue, specificpromoter.

The host cells used to express the molecules identified by the methodsof the invention may be either bacterial cells such as Escherichia coli,or, preferably, eukaryotic cells, especially for the expression of wholerecombinant immunoglobulin molecule. In particular, mammalian cells suchas Chinese hamster ovary cells (CHO), in conjunction with a vector suchas the major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for immunoglobulins(Foecking et al., 1998, Gene 45:101; Cockett et al., 1990,Bio/Technology 8:2).

A variety of host-expression vector systems may be utilized to expressthe molecules identified by the methods of the invention. Suchhost-expression systems represent vehicles by which the coding sequencesof the molecules of the invention may be produced and subsequentlypurified, but also represent cells which may, when transformed ortransfected with the appropriate nucleotide coding sequences, expressthe molecules of the invention in situ. These include, but are notlimited to, microorganisms such as bacteria (e.g., E. coli and B.subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA orcosmid DNA expression vectors containing coding sequences for themolecules identified by the methods of the invention; yeast (e.g.,Saccharomyces Pichia) transformed with recombinant yeast expressionvectors containing sequences encoding the molecules identified by themethods of the invention; insect cell systems infected with recombinantvirus expression vectors (e.g., baculovirus) containing the sequencesencoding the molecules identified by the methods of the invention; plantcell systems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing sequences encoding the molecules identified by themethods of the invention; or mammalian cell systems (e.g., COS, CHO,BHK, 293, 293T, 3T3 cells), lymphotic cells (see U.S. Pat. No.5,807,715), Per C.6 cells (human retinal cells developed by Crucell)harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the moleculebeing expressed. For example, when a large quantity of such a protein isto be produced, for the generation of pharmaceutical compositions of anantibody, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include, but are not limited, to the E. coli expression vectorpUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the antibodycoding sequence may be ligated individually into the vector in framewith the lac Z coding region so that a fusion protein is produced; pINvectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; VanHeeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEXvectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption and binding to a matrix glutathione-agarose beads followed byelution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (e.g., the polyhedrin gene) ofthe virus and placed under control of an AcNPV promoter (e.g., thepolyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the immunoglobulin molecule in infected hosts (e.g., seeLogan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., 1987,Methods in Enzymol. 153:51-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 andHs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably express anantibody of the invention may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express theantibodies of the invention. Such engineered cell lines may beparticularly useful in screening and evaluation of compounds thatinteract directly or indirectly with the antibodies of the invention.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48: 202), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78: 1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12: 488-505; Wu and Wu, 1991, 3:87-95; Tolstoshev,1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem.62:191-217; May, 1993, TIB TECH 11(5):155-215). Methods commonly knownin the art of recombinant DNA technology which can be used are describedin Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology,John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, ALaboratory Manual, Stockton Press, NY; and in Chapters 12 and 13,Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, JohnWiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1;and hygro, which confers resistance to hygromycin (Santerre et al.,1984, Gene 30:147).

The expression levels of an antibody of the invention can be increasedby vector amplification (for a review, see Bebbington and Hentschel, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, NewYork, 1987). When a marker in the vector system expressing an antibodyis amplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the nucleotide sequence of theantibody, production of the antibody will also increase (Crouse et al.,1983, Mol. Cell. Biol. 3:257).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain should be placedbefore the heavy chain to avoid an excess of toxic free heavy chain(Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci.USA 77:2197). The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA.

Once a molecule of the invention has been recombinantly expressed, itmay be purified by any method known in the art for purification ofpolypeptides or antibodies, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of polypeptides or antibodies.

5.4 Prophylactic and Therapeutic Methods

The molecules of the invention with conferred effector function activityare particularly useful for the treatment and/or prevention of adisease, disorder or infection where an enhanced efficacy of effectorcell function (e.g., ADCC) mediated by FcγR is desired (e.g., cancer,infectious disease), and in enhancing the therapeutic efficacy oftherapeutic antibodies, the effect of which is mediated by an effectorfunction activity, e.g., ADCC.

The invention encompasses methods and compositions for treatment,prevention or management of a cancer in a subject, comprisingadministering to the subject a therapeutically effective amount of oneor more molecules comprising a variant Fc region engineered inaccordance with the invention, which molecule further binds a cancerantigen. Molecules of the invention comprising the variant Fc regionsare particularly useful for the prevention, inhibition, reduction ofgrowth or regression of primary tumors, metastasis of cancer cells, andinfectious diseases. Although not intending to be bound by a particularmechanism of action, molecules of the invention enhance the efficacy ofcancer therapeutics by enhancing antibody mediated effector functionresulting in an enhanced rate of tumor clearance or an enhanced rated oftumor reduction or a combination thereof. In alternate embodiments, themodified antibodies of the invention enhance the efficacy of cancertherapeutics by conferring oligomerization activity to variant Fcregion, resulting in cross-linking of cell surface antigens and/orreceptors and enhanced apoptosis or negative growth regulatorysignaling.

According to an aspect of the invention, immunotherapeutics may beenhanced by modifying the Fc region in accordance with the invention toconfer or increase the potency of an antibody effector functionactivity, e.g., ADCC, CDC, phagocytosis, opsonization, etc., of theimmunotherapeutic. In a specific embodiment, antibody dependent cellulartoxicity and/or phagocytosis of tumor cells or infected cells isenhanced by modifying immunotherapeutics with variant Fc regions of theinvention. Molecules of the invention may enhance the efficacy ofimmunotherapy treatment by enhancing at least one antibody-mediatedeffector function activity. In one particular embodiment, the efficacyof immunotherapy treatment is enhanced by enhancing the complementdependent cascade. In another embodiment of the invention, the efficacyof immunotherapy treatment is enhanced by enhancing the phagocytosisand/or opsonization of the targeted cells, e.g., tumor cells. In anotherembodiment of the invention, the efficacy of treatment is enhanced byenhancing antibody-dependent cell-mediated cytotoxicity (“ADCC”) indestruction of the targeted cells, e.g., tumor cells. The molecules ofthe invention may make an antibody that does not have a therapeuticeffect in patients or in a subpopulation of patients have a therapeuticeffect.

Although not intending to be bound by a particular mechanism of action,therapeutic antibodies engineered in accordance with the invention haveenhanced therapeutic efficacy, in part, due to the ability of the Fcportion of the antibody to bind a target cell which expresses theparticular FcγRs at reduced levels, for example, by virtue of theability of the antibody to remain on the target cell longer due to animproved off rate for FcγR interaction.

The antibodies of the invention with enhanced affinity and avidity forFcγRs are particularly useful for the treatment, prevention ormanagement of a cancer, or another disease or disorder, in a subject,wherein the FcγRs are expressed at low levels in the target cellpopulations. As used herein, FcγR expression in cells is defined interms of the density of such molecules per cell as measured using commonmethods known to those skilled in the art. The molecules of theinvention comprising variant Fc regions preferably also have a conferredor an enhanced avidity and affinity and/or effector function in cellswhich express a target antigen, e.g., a cancer antigen, at a density of30,000 to 20,000 molecules/cell, at a density of 20,000 to 10,000molecules/cell, at a density of 10,000 molecules/cell or less, at adensity of 5000 molecules/cell or less, or at a density of 1000molecules/cell or less. The molecules of the invention have particularutility in treatment, prevention or management of a disease or disorder,such as cancer, in a sub-population, wherein the target antigen isexpressed at low levels in the target cell population.

The molecules of the invention may also be advantageously utilized incombination with other therapeutic agents known in the art for thetreatment or prevention of diseases, such as cancer, autoimmune disease,inflammatory disorders, and infectious diseases. In a specificembodiment, molecules of the invention may be used in combination withmonoclonal or chimeric antibodies, lymphokines, or hematopoietic growthfactors (such as, e.g., IL-2, IL-3 and IL-7), which, for example, serveto increase the number or activity of effector cells which interact withthe molecules and, increase immune response. The molecules of theinvention may also be advantageously utilized in combination with one ormore drugs used to treat a disease, disorder, or infection such as, forexample anti-cancer agents, anti-inflammatory agents or anti-viralagents, e.g., as detailed in sections 5.4.1.2 and 5.4.2.1 below.

5.4.1 Cancers

The invention encompasses methods and compositions for treatment orprevention of cancer in a subject comprising administering to thesubject a therapeutically effective amount of one or more moleculescomprising a variant Fc region. In some embodiments, the inventionencompasses methods and compositions for the treatment or prevention ofcancer in a subject with FcγR polymorphisms such as those homozygous forthe FyRIIIA-158V or FcγRIIIA-158F alleles. In some embodiments, theinvention encompasses engineering therapeutic antibodies, e.g., tumorspecific monoclonal antibodies in accordance with the methods of theinvention such that the engineered antibodies have enhanced efficacy inpatients homozygous for the low affinity allele of FcγRIIIA (158F). Inother embodiments, the invention encompasses engineering therapeuticantibodies, e.g., tumor specific monoclonal antibodies in accordancewith the methods of the invention such that the engineered antibodieshave enhanced efficacy in patients homozygous for the high affinityallele of FcγRIIIA (158V).

The efficacy of monoclonal antibodies may depend on the FcγRpolymorphism of the subject (Carton et al., 2002 Blood, 99: 754-8; Wenget al., 2003 J Clin Oncol. 21(21):3940-7 both of which are incorporatedherein by reference in their entireties). These receptors are expressedon the surface of the effector cells and mediate ADCC. High affinityalleles, of the low affinity activating receptors, improve the effectorcells' ability to mediate ADCC. The methods of the invention allowengineering molecules harboring Fc mutations to enhance their affinityto FcγR on effector cells via their altered Fc domains. The engineeredantibodies of the invention provide better immunotherapy reagents forpatients regardless of their FcγR polymorphism.

Molecules harboring the Fc variants are tested by ADCC using either acultured cell line or patient derived PMBC cells to determine theability of the Fc mutations to enhance ADCC. Standard ADCC is performedusing methods disclosed herein. Lymphocytes are harvested fromperipheral blood using a Ficoll-Paque gradient (Pharmacia). Targetcells, i.e., cultured cell lines or patient derived cells, are loadedwith Europium (PerkinElmer) and incubated with effectors for 4 hrs at37° C. Released Europium is detected using a fluorescent plate reader(Wallac). The resulting ADCC data indicates the efficacy of the Fcvariants to trigger NK cell mediated cytotoxicity and establish which Fcvariants can be tested with both patient samples and elutriatedmonocytes. Fc variants showing the greatest potential for enhancing theefficacy of the molecule are then tested in an ADCC assay using PBMCsfrom patients. PBMC from healthy donors are used as effector cells.

According to an aspect of the invention, molecules of the inventioncomprising variant Fc regions enhance the efficacy of immunotherapy byconferring or increasing the potency of an antibody effector functionrelative to a molecule containing the wild-type Fc region, e.g., ADCC,CDC, phagocytosis, opsonization, etc. In a specific embodiment, antibodydependent cellular toxicity and/or phagocytosis of tumor cells isconferred or enhanced using the molecules of the invention with variantFc regions. Molecules of the invention may enhance the efficacy ofimmunotherapy cancer treatment by conferring or enhancing at least oneantibody-mediated effector function. In one particular embodiment, amolecule of the invention comprising a variant Fc region confers orenhances the efficacy of immunotherapy treatment by enhancing thecomplement dependent cascade. In another embodiment of the invention,the molecule of the invention comprising a variant Fc region enhancesthe efficacy of immunotherapy treatment by conferring or enhancing thephagocytosis and/or opsonization of the targeted tumor cells. In anotherembodiment of the invention, the molecule of the invention comprising avariant Fc region enhances the efficacy of treatment by conferring orenhancing antibody-dependent cell-mediated cytotoxicity (“ADCC”) indestruction of the targeted tumor cells.

The invention further contemplates engineering therapeutic antibodies(e.g., tumor specific monoclonal antibodies) for enhancing thetherapeutic efficacy of the therapeutic antibody, for example, byenhancing the effector function of the therapeutic antibody (e.g.,ADCC), or conferring effector function to a therapeutic antibody whichdoesn't have that effector function (at least detectable in an in vitroor in vivo assay). Preferably the therapeutic antibody is a cytotoxicand/or opsonizing antibody. It will be appreciated by one of skill inthe art, that once molecules of the invention with desired bindingproperties (e.g., molecules with variant Fc regions with at least oneamino acid modification, which modification enhances the affinity of thevariant Fc region for FcγRIIIA and/or FcγRIIA relative to a comparablemolecule, comprising a wild-type Fc region) have been identified (SeeSection 5.2 and Table 8) according to the methods of the invention,therapeutic antibodies may be engineered using standard recombinant DNAtechniques and any known mutagenesis techniques, as described in Section5.2.2 to produce engineered therapeutic carrying the identified mutationsites with the desired binding properties. Any of the therapeuticantibodies listed in Table 11 that have demonstrated therapeutic utilityin cancer treatment, may be engineered according to the methods of theinvention, for example, by modifying the Fc region to confer an effectorfunction or have an enhanced affinity for FcγRIIIA and/or FcγRIIAcompared to a therapeutic antibody having a wild-type Fc region.

The Fc variants of the invention may be incorporated into therapeuticantibodies such as those disclosed herein or other Fc fusion clinicalcandidates, i.e., a molecule comprising an Fc regions which has beenapproved for us in clinical trials or any other molecule that maybenefit from the Fc variants of the instant invention, humanized,affinity matured, modified or engineered versions thereof.

The invention also encompasses engineering any other polypeptidecomprising an Fc region which has therapeutic utility, including but notlimited to ENBREL, according to the methods of the invention, in orderto enhance the therapeutic efficacy of such polypeptides, for example,by enhancing the effector function of the polypeptide comprising an Fcregion.

TABLE 11 THERAPEUTIC ANTIBODIES THAT CAN BE ENGINEERED ACCORDING TO THEMETHODS OF THE INVENTION Company Product Disease Target AbgenixABX-EGF ™ anti-EGF Cancer EGF receptor Receptor Antibody AltaRexOvaRex ™ anti-CA125 ovarian cancer tumor antigen CA125 AntibodyBravaRex ™ anti-MUC1 metastatic tumor antigen MUC1 Antibody cancersAntisoma Theragyn ™ ovarian cancer PEM antigen (pemtumomabytrrium- 90)Therex ™ breast cancer PEM antigen Boehringer Blvatuzumab head & neckCD44 Ingelheim cancer Centocor/J&J Panorex ™ anti-17-1A Colorectal 17-1AAntibody cancer ReoPro ™ PTCA gp IIIb/IIIa ReoPro ™ Acute MI gpIIIb/IIIa ReoPro ™ Ischemic stroke gp IIIb/IIIa Corixa Bexocar ™ NHLCD20 CRC MAb, idiotypic 105AD7 colorectal cancer gp72 Technology vaccineCrucell Anti-EpCAM ™ cancer Ep-CAM Cytoclonal MAb, lung cancer non-smallcell NA lung cancer Genentech Herceptin ™ anti-Her-2 metastatic breastHER-2 Antibody cancer Herceptin ™ anti-Her-2 early stage HER-2 Antibodybreast cancer Rituxan ™ anti-CD20 Relapsed/refractory CD20 Antibodylow-grade or follicular NHL Rituxan ™ anti-CD20 intermediate & CD20Antibody high-grade NHL MAb-VEGF NSCLC, VEGF metastatic MAb-VEGFColorectal VEGF cancer, metastatic AMD Fab age-related CD18 maculardegeneration E-26 (2^(nd) gen. IgE) allergic asthma IgE & rhinitis IDECZevalin ™ (Rituxan ™ low grade of CD20 anti-CD20 Antibody + follicular,yttrium-90) relapsed or refractory, CD20-positive, B-cell NHL andRituximab- refractory NHL ImClone Cetuximab + innotecan refractory EGFreceptor colorectal carcinoma Cetuximab + cisplatin & newly diagnosedEGF receptor radiation or recurrent head & neck cancer Cetuximab + newlydiagnosed EGF receptor gemcitabine metastatic pancreatic carcinomaCetuximab + cisplatin + recurrent or EGF receptor 5FU or Taxolmetastatic head & neck cancer Cetuximab + newly diagnosed EGF receptorcarboplatin + paclitaxel non-small cell lung carcinoma Cetuximab +cisplatin head & neck EGF receptor cancer (extensive incurable local-regional disease & distant metasteses) Cetuximab + radiation locallyadvanced EGF receptor head & neck carcinoma BEC2 + Bacillus small celllung mimics ganglioside Calmette Guerin carcinoma GD3 BEC2 + Bacillusmelanoma mimics ganglioside Calmette Guerin GD3 IMC-1C11 colorectalcancer VEGF-receptor with liver metasteses ImmonoGen nuC242-DM1Colorectal, nuC242 gastric, and pancreatic cancer ImmunoMedicsLymphoCide ™ anti- Non-Hodgkins CD22 CD22 Antibody lymphoma LymphoCideY-90 ™ Non-Hodgkins CD22 anti-CD22 Antibody lymphoma CEA-Cide ™metastatic solid CEA tumors CEA-Cide ™ Y-90 metastatic solid CEA tumorsCEA-Scan ™ (Tc-99m- colorectal cancer CEA labeled arcitumomab)(radioimaging) CEA-Scan ™ (Tc-99m- Breast cancer CEA labeledarcitumomab) (radioimaging) CEA-Scan ™ (Tc-99m- lung cancer CEA labeledarcitumomab) (radioimaging) CEA-Scan ™ (Tc-99m- intraoperative CEAlabeled arcitumomab) tumors (radio imaging) LeukoScan ™ (Tc-99m- softtissue CEA labeled sulesomab) infection (radioimaging) LymphoScan ™ (Tc-lymphomas CD22 99m-labeled) (radioimaging) AFP-Scan ™ (Tc-99m- liver 7gem-cell AFP labeled) cancers (radioimaging) Intracel HumaRAD-HN ™ (+head & neck NA yttrium-90) cancer HumaSPECT ™ colorectal NA imagingMedarex MDX-101 ™ (CTLA-4) Prostate and CTLA-4 other cancers MDX-210 ™(her-2 Prostate cancer HER-2 overexpression) MDX-210 ™/MAK Cancer HER-2MedImmune Vitaxin ™ Cancer αvβ₃ Merck KGaA MAb 425 Various cancers EGFreceptor IS-IL-2 Various cancers Ep-CAM Millennium Campath ® anti-CD52chronic CD52 Antibody lymphocytic (alemtuzumab) leukemia NeoRxCD20-streptavidin (+ Non-Hodgkins CD20 biotin-yttrium 90) lymphomaAvidicin ™ (albumin + metastatic NA NRLU13) cancer Peregrine Oncolym ™(+ iodine- Non-Hodgkins HLA-DR 10 beta 131) lymphoma Cotara ™ (+iodine-131) unresectable DNA-associated malignant proteins gliomaPharmacia C215 ™ (+ pancreatic NA Corporation staphylococcal cancerenterotoxin) MAb, lung/kidney lung & kidney NA cancer cancer nacolomabtafenatox colon & NA (C242 + staphylococcal pancreatic enterotoxin)cancer Protein Design Nuvion ™ T cell CD3 Labs malignancies SMART M195 ™anti- AML CD33 CD33 Antibody SMART 1D10 ™ anti- NHL HLA-DR antigenHLA-DR Antibody Titan CEAVac ™ colorectal CEA cancer, advanced TriGem ™metastatic GD2-ganglioside melanoma & small cell lung cancer TriAb ™metastatic breast MUC-1 cancer Trilex CEAVa ™ c colorectal CEA cancer,advanced TriGem ™ metastatic GD2-ganglioside melanoma & small cell lungcancer TriAb ™ metastatic breast MUC-1 cancer Viventia NovoMAb-G2 ™Non-Hodgkins NA Biotech radiolabeled lymphoma Monopharm C ™ colorectal &SK-1 antigen pancreatic carcinoma GlioMAb-H ™ (+ gliorna, NA gelonintoxin) melanoma & neuroblastoma Xoma Rituxan ™ anti-CD20Relapsed/refractory CD20 Antibody low-grade or follicular NHL Rituxan ™anti-CD20 intermediate & CD20 Antibody high-grade NHL ING-1 ™adenomcarcinoma Ep-CAM

Accordingly, the invention provides methods of preventing or treatingcancer characterized by a cancer antigen, using a therapeutic antibodythat binds a cancer antigen and is cytotoxic and has been modified atone or more sites in the Fc region, according to the invention, to bindFcγRIIIA and/or FcγRIIA with a higher affinity than the parenttherapeutic antibody, and/or mediates one or more effector functions(e.g., ADCC, phagocytosis) either not detectably mediated by the parentantibody or more effectively than the parent antibody. In anotherembodiment, the invention provides methods of preventing or treatingcancer characterized by a cancer antigen, using a therapeutic antibodythat binds a cancer antigen and is cytotoxic, and has been engineeredaccording to the invention to bind FcγRIIIA and/or FcγRIIA with a higheraffinity and bind FcγRIIB with a lower affinity than the parenttherapeutic antibody, and/or mediates one or more effector functions(e.g., ADCC, phagocytosis) either not detectably mediated by the parentantibody or more effectively than the parent antibody. The therapeuticantibodies that have been engineered according to the invention areuseful for prevention or treatment of cancer, since they have anenhanced cytotoxic activity (e.g., enhanced tumor cell killing and/orenhanced for example, ADCC activity or CDC activity).

Cancers associated with a cancer antigen may be treated or prevented byadministration of a therapeutic antibody that binds a cancer antigen andis cytotoxic, and has been engineered according to the methods of theinvention to have, for example, a conferred or an enhanced effectorfunction. In one particular embodiment, the therapeutic antibodiesengineered according to the methods of the invention enhance theantibody-mediated cytotoxic effect of the antibody directed at theparticular cancer antigen. For example, but not by way of limitation,cancers associated with the following cancer antigens may be treated orprevented by the methods and compositions of the invention: KS 1/4pan-carcinoma antigen (Perez and Walker, 1990, J. Immunol. 142:32-37;Bumal, 1988, Hybridoma 7(4):407-415), ovarian carcinoma antigen (CA125)(Yu et al., 1991, Cancer Res. 51(2):48-475), prostatic acid phosphate(Tailor et al., 1990, Nucl. Acids Res. 18(1):4928), prostate specificantigen (Henttu and Vihko, 1989, Biochem. Biophys. Res. Comm.10(2):903-910; Israeli et al., 1993, Cancer Res. 53:227-230),melanoma-associated antigen p97 (Estin et al., 1989, J. Natl. CancerInstil. 81(6):445-44), melanoma antigen gp75 (Vijayasardahl et al.,1990, J. Exp. Med. 171(4):1375-1380), high molecular weight melanomaantigen (HMW-MAA) (Natali et al., 1987, Cancer 59:55-3; Mittelman etal., 1990, J. Clin. Invest. 86:2136-2144)), prostate specific membraneantigen, carcinoembryonic antigen (CEA) (Foon et al., 1994, Proc. Am.Soc. Clin. Oncol. 13:294), polymorphic epithelial mucin antigen, humanmilk fat globule antigen, Colorectal tumor-associated antigens such as:CEA, TAG-72 (Yokata et al., 1992, Cancer Res. 52:3402-3408), C017-1A(Ragnhammar et al., 1993, Int. J. Cancer 53:751-758); GICA 19-9 (Herlynet al., 1982, J. Clin. Immunol. 2:135), CTA-1 and LEA, Burkitt'slymphoma antigen-38.13, CD19 (Ghetie et al., 1994, Blood 83:1329-1336),human B-lymphoma antigen-CD20 (Reff et al., 1994, Blood 83:435-445),CD33 (Sgouros et al., 1993, J. Nucl. Med. 34:422-430), melanoma specificantigens such as ganglioside GD2 (Saleh et al., 1993, J. Immunol., 151,3390-3398), ganglioside GD3 (Shitara et al., 1993, Cancer Immunol.Immunother. 36:373-380), ganglioside GM2 (Livingston et al., 1994, J.Clin. Oncol. 12:1036-1044), ganglioside GM3 (Hoon et al., 1993, CancerRes. 53:5244-5250), tumor-specific transplantation type of cell-surfaceantigen (TSTA) such as virally-induced tumor antigens includingT-antigen DNA tumor viruses and envelope antigens of RNA tumor viruses,oncofetal antigen-alpha-fetoprotein such as CEA of colon, bladder tumoroncofetal antigen (Hellstrom et al., 1985, Cancer. Res. 45:2210-2188),differentiation antigen such as human lung carcinoma antigen L6, L20(Hellstrom et al., 1986, Cancer Res. 46:3917-3923), antigens offibrosarcoma, human leukemia T cell antigen-Gp37(Bhattacharya-Chatterjee et al., 1988, J. of Immun. 141:1398-1403),neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR(Epidermal growth factor receptor), HER2 antigen (p185^(HER2)),polymorphic epithelial mucin (PEM) (Hilkens et al., 1992, Trends in Bio.Chem. Sci. 17:359), malignant human lymphocyte antigen-APO-1 (Bernhardet al., 1989, Science 245:301-304), differentiation antigen (Feizi,1985, Nature 314:53-57) such as I antigen found in fetal erthrocytes andprimary endoderm, I(Ma) found in gastric adencarcinomas, M18 and M39found in breast epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9,Myl, VIM-D5, and D₁56-22 found in colorectal cancer, TRA-1-85 (bloodgroup H), C14 found in colonic adenocarcinoma, F3 found in lungadenocarcinoma, AH6 found in gastric cancer, Y hapten, Le found inembryonal carcinoma cells, TL5 (blood group A), EGF receptor found inA431 cells, E₁ series (blood group B) found in pancreatic cancer, FC10.2found in embryonal carcinoma cells, gastric adenocarcinoma, CO-514(blood group Le^(a)) found in adenocarcinoma, NS-10 found inadenocarcinomas, CO-43 (blood group Le^(b)), G49, EGF receptor, (bloodgroup ALe^(b)/LeY) found in colonic adenocarcinoma, 19.9 found in coloncancer, gastric cancer mucins, T₅A₇ found in myeloid cells, R₂₄ found inmelanoma, 4.2, G_(D3), D1.1, OFA-1, G_(M2), OFA-2, G_(D2), M1:22:25:8found in embryonal carcinoma cells and SSEA-3, SSEA-4 found in 4-8-cellstage embryos. In another embodiment, the antigen is a T cell receptorderived peptide from a cutaneous T cell lymphoma (see Edelson, 1998, TheCancer Journal 4:62).

Cancers and related disorders that can be treated or prevented bymethods and compositions of the present invention include, but are notlimited to, the following: Leukemias including, but not limited to,acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemiassuch as myeloblastic, promyelocytic, myelomonocytic, monocytic,erythroleukemia leukemias and myelodysplastic syndrome, chronicleukemias such as but not limited to, chronic myelocytic (granulocytic)leukemia, chronic lymphocytic leukemia, hairy cell leukemia;polycythemia vera; lymphomas such as but not limited to Hodgkin'sdisease, non-Hodgkin's disease; multiple myelomas such as but notlimited to smoldering multiple myeloma, nonsecretory myeloma,osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma andextramedullary plasmacytoma; Waldenström's macroglobulinemia; monoclonalgammopathy of undetermined significance; benign monoclonal gammopathy;heavy chain disease; bone and connective tissue sarcomas such as but notlimited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma,malignant giant cell tumor, fibrosarcoma of bone, chordoma, periostealsarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma;brain tumors including but not limited to, glioma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, nonglial tumor, acousticneurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, primary brain lymphoma; breast cancer including, but notlimited to, adenocarcinoma, lobular (small cell) carcinoma, intraductalcarcinoma, medullary breast cancer, mucinous breast cancer, tubularbreast cancer, papillary breast cancer, Paget's disease, andinflammatory breast cancer; adrenal cancer, including but not limitedto, pheochromocytom and adrenocortical carcinoma; thyroid cancer such asbut not limited to papillary or follicular thyroid cancer, medullarythyroid cancer and anaplastic thyroid cancer; pancreatic cancer,including but not limited to, insulinoma, gastrinoma, glucagonoma,vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor;pituitary cancers including but not limited to, Cushing's disease,prolactin-secreting tumor, acromegaly, and diabetes insipius; eyecancers including but not limited to, ocular melanoma such as irismelanoma, choroidal melanoma, and cilliary body melanoma, andretinoblastoma; vaginal cancers, including but not limited to, squamouscell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, includingbut not limited to, squamous cell carcinoma, melanoma, adenocarcinoma,basal cell carcinoma, sarcoma, and Paget's disease; cervical cancersincluding but not limited to, squamous cell carcinoma, andadenocarcinoma; uterine cancers including but not limited to,endometrial carcinoma and uterine sarcoma; ovarian cancers including butnot limited to, ovarian epithelial carcinoma, borderline tumor, germcell tumor, and stromal tumor; esophageal cancers including but notlimited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma,mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma,plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma;stomach cancers including but not limited to, adenocarcinoma, fungating(polypoid), ulcerating, superficial spreading, diffusely spreading,malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; coloncancers; rectal cancers; liver cancers including but not limited tohepatocellular carcinoma and hepatoblastoma, gallbladder cancersincluding but not limited to, adenocarcinoma; cholangiocarcinomasincluding but not limited to, pappillary, nodular, and diffuse; lungcancers including but not limited to, non-small cell lung cancer,squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma,large-cell carcinoma and small-cell lung cancer; testicular cancersincluding but not limited to, germinal tumor, seminoma, anaplastic,classic (typical), spermatocytic, nonseminoma, embryonal carcinoma,teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancersincluding but not limited to, adenocarcinoma, leiomyosarcoma, andrhabdomyosarcoma; penal cancers; oral cancers including but not limitedto, squamous cell carcinoma; basal cancers; salivary gland cancersincluding but not limited to, adenocarcinoma, mucoepidermoid carcinoma,and adenoidcystic carcinoma; pharynx cancers including but not limitedto, squamous cell cancer, and verrucous; skin cancers including but notlimited to, basal cell carcinoma, squamous cell carcinoma and melanoma,superficial spreading melanoma, nodular melanoma, lentigo malignantmelanoma, acral lentiginous melanoma; kidney cancers including but notlimited to, renal cell cancer, adenocarcinoma, hypernephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer);Wilms' tumor; bladder cancers including but not limited to, transitionalcell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. Inaddition, cancers include myxosarcoma, osteogenic sarcoma,endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma,hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogeniccarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillarycarcinoma and papillary adenocarcinomas (for a review of such disorders,see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co.,Philadelphia and Murphy et al., 1997, Informed Decisions: The CompleteBook of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin,Penguin Books U.S.A., Inc., United States of America).

Accordingly, the methods and compositions of the invention are alsouseful in the treatment or prevention of a variety of cancers or otherabnormal proliferative diseases, including (but not limited to) thefollowing: carcinoma, including that of the bladder, breast, colon,kidney, liver, lung, ovary, pancreas, stomach, prostate, cervix, thyroidand skin; including squamous cell carcinoma; hematopoietic tumors oflymphoid lineage, including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkettslymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias and promyelocytic leukemia; tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; othertumors, including melanoma, seminoma, tetratocarcinoma, neuroblastomaand glioma; tumors of the central and peripheral nervous system,including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors ofmesenchymal origin, including fibrosafcoma, rhabdomyoscarama, andosteosarcoma; and other tumors, including melanoma, xenodermapegmentosum, keratoactanthoma, seminoma, thyroid follicular cancer andteratocarcinoma. It is also contemplated that cancers caused byaberrations in apoptosis would also be treated by the methods andcompositions of the invention. Such cancers may include but not belimited to follicular lymphomas, carcinomas with p53 mutations, hormonedependent tumors of the breast, prostate and ovary, and precancerouslesions such as familial adenomatous polyposis, and myelodysplasticsyndromes. In specific embodiments, malignancy or dysproliferativechanges (such as metaplasias and dysplasias), or hyperproliferativedisorders, are treated or prevented by the methods and compositions ofthe invention in the ovary, bladder, breast, colon, lung, skin,pancreas, or uterus. In other specific embodiments, sarcoma, melanoma,or leukemia is treated or prevented by the methods and compositions ofthe invention.

In a specific embodiment, a molecule of the invention (e.g., an antibodycomprising a variant Fc region, or a therapeutic monoclonal antibodyengineered according to the methods of the invention) inhibits orreduces the growth of cancer cells by at least 99%, at least 95%, atleast 90%, at least 85%, at least 80%, at least 75%, at least 70%, atleast 60%, at least 50%, at least 45%, at least 40%, at least 45%, atleast 35%, at least 30%, at least 25%, at least 20%, or at least 10%relative to the growth of cancer cells in the absence of said moleculeof the invention.

In a specific embodiment, a molecule of the invention (e.g., an antibodycomprising a variant Fc region, or a therapeutic monoclonal antibodyengineered according to the methods of the invention) kills cells orinhibits or reduces the growth of cancer cells at least 5%, at least10%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 60%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least100% better than the parent molecule.

5.4.1.1 Combination Therapy

The invention further encompasses administering the molecules of theinvention in combination with other therapies known to those skilled inthe art for the treatment or prevention of cancer or infectious disease,including but not limited to, current standard and experimentalchemotherapies, hormonal therapies, biological therapies,immunotherapies, radiation therapies, or surgery. In some embodiments,the molecules of the invention may be administered in combination with atherapeutically or prophylactically effective amount of one or moreanti-cancer agents, therapeutic antibodies (e.g., antibodies listed inTable 11), or other agents known to those skilled in the art for thetreatment and/or prevention of cancer (See Section 5.4.1.2).

In certain embodiments, one or more molecule of the invention isadministered to a mammal, preferably a human, concurrently with one ormore other therapeutic agents useful for the treatment of cancer. Theterm “concurrently” is not limited to the administration of prophylacticor therapeutic agents at exactly the same time, but rather it is meantthat a molecule of the invention and the other agent are administered toa mammal in a sequence and within a time interval such that the moleculeof the invention can act together with the other agent to provide anincreased benefit than if they were administered otherwise. For example,each prophylactic or therapeutic agent (e.g., chemotherapy, radiationtherapy, hormonal therapy or biological therapy) may be administered atthe same time or sequentially in any order at different points in time;however, if not administered at the same time, they should beadministered sufficiently close in time so as to provide the desiredtherapeutic or prophylactic effect. Each therapeutic agent can beadministered separately, in any appropriate form and by any suitableroute. In various embodiments, the prophylactic or therapeutic agentsare administered less than 1 hour apart, at about 1 hour apart, at about1 hour to about 2 hours apart, at about 2 hours to about 3 hours apart,at about 3 hours to about 4 hours apart, at about 4 hours to about 5hours apart, at about 5 hours to about 6 hours apart, at about 6 hoursto about 7 hours apart, at about 7 hours to about 8 hours apart, atabout 8 hours to about 9 hours apart, at about 9 hours to about 10 hoursapart, at about 10 hours to about 11 hours apart, at about 11 hours toabout 12 hours apart, no more than 24 hours apart or no more than 48hours apart. In preferred embodiments, two or more components areadministered within the same patient visit.

In other embodiments, the prophylactic or therapeutic agents areadministered at about 2 to 4 days apart, at about 4 to 6 days apart, atabout 1 week part, at about 1 to 2 weeks apart, or more than 2 weeksapart. In preferred embodiments, the prophylactic or therapeutic agentsare administered in a time frame where both agents are still active. Oneskilled in the art would be able to determine such a time frame bydetermining the half life of the administered agents.

In certain embodiments, the prophylactic or therapeutic agents of theinvention are cyclically administered to a subject. Cycling therapyinvolves the administration of a first agent for a period of time,followed by the administration of a second agent and/or third agent fora period of time and repeating this sequential administration. Cyclingtherapy can reduce the development of resistance to one or more of thetherapies, avoid or reduce the side effects of one of the therapies,and/or improves the efficacy of the treatment.

In certain embodiments, prophylactic or therapeutic agents areadministered in a cycle of less than about 3 weeks, about once every twoweeks, about once every 10 days or about once every week. One cycle cancomprise the administration of a therapeutic or prophylactic agent byinfusion over about 90 minutes every cycle, about 1 hour every cycle,about 45 minutes every cycle. Each cycle can comprise at least 1 week ofrest, at least 2 weeks of rest, at least 3 weeks of rest. The number ofcycles administered is from about 1 to about 12 cycles, more typicallyfrom about 2 to about 10 cycles, and more typically from about 2 toabout 8 cycles.

In yet other embodiments, the therapeutic and prophylactic agents of theinvention are administered in metronomic dosing regimens, either bycontinuous infusion or frequent administration without extended restperiods. Such metronomic administration can involve dosing at constantintervals without rest periods. Typically the therapeutic agents, inparticular cytotoxic agents, are used at lower doses. Such dosingregimens encompass the chronic daily administration of relatively lowdoses for extended periods of time. In preferred embodiments, the use oflower doses can minimize toxic side effects and eliminate rest periods.In certain embodiments, the therapeutic and prophylactic agents aredelivered by chronic low-dose or continuous infusion ranging from about24 hours to about 2 days, to about 1 week, to about 2 weeks, to about 3weeks to about 1 month to about 2 months, to about 3 months, to about 4months, to about 5 months, to about 6 months. The scheduling of suchdose regimens can be optimized by the skilled oncologist.

In other embodiments, courses of treatment are administered concurrentlyto a mammal, i.e., individual doses of the therapeutics are administeredseparately yet within a time interval such that molecules of theinvention can work together with the other agent or agents. For example,one component may be administered one time per week in combination withthe other components that may be administered one time every two weeksor one time every three weeks. In other words, the dosing regimens forthe therapeutics are carried out concurrently even if the therapeuticsare not administered simultaneously or within the same patient visit.

When used in combination with other prophylactic and/or therapeuticagents, the molecules of the invention and the prophylactic and/ortherapeutic agent can act additively or, more preferably,synergistically. In one embodiment, a molecule of the invention isadministered concurrently with one or more therapeutic agents in thesame pharmaceutical composition. In another embodiment, a molecule ofthe invention is administered concurrently with one or more othertherapeutic agents in separate pharmaceutical compositions. In stillanother embodiment, a molecule of the invention is administered prior toor subsequent to administration of another prophylactic or therapeuticagent. The invention contemplates administration of a molecule of theinvention in combination with other prophylactic or therapeutic agentsby the same or different routes of administration, e.g., oral andparenteral. In certain embodiments, when a molecule of the invention isadministered concurrently with another prophylactic or therapeutic agentthat potentially produces adverse side effects including, but notlimited to, toxicity, the prophylactic or therapeutic agent canadvantageously be administered at a dose that falls below the thresholdthat the adverse side effect is elicited.

The dosage amounts and frequencies of administration provided herein areencompassed by the terms therapeutically effective and prophylacticallyeffective. The dosage and frequency further will typically varyaccording to factors specific for each patient depending on the specifictherapeutic or prophylactic agents administered, the severity and typeof cancer, the route of administration, as well as age, body weight,response, and the past medical history of the patient. Suitable regimenscan be selected by one skilled in the art by considering such factorsand by following, for example, dosages reported in the literature andrecommended in the Physician's Desk Reference (56^(th) ed., 2002).

5.4.1.2 Other Therapeutic/Prophylactic Agents

In a specific embodiment, the methods of the invention encompass theadministration of one or more molecules of the invention with one ormore therapeutic agents used for the treatment and/or prevention ofcancer. In one embodiment, angiogenesis inhibitors may be administeredin combination with the molecules of the invention. Angiogenesisinhibitors that can be used in the methods and compositions of theinvention include but are not limited to: Angiostatin (plasminogenfragment); antiangiogenic antithrombin III; Angiozyme; ABT-627; Bay12-9566; Benefin; Bevacizumab; BMS-275291; cartilage-derived inhibitor(CDI); CAI; CD59 complement fragment; CEP-7055; Col 3; CombretastatinA-4; Endostatin (collagen XVIII fragment); Fibronectin fragment;Gro-beta; Halofuginone; Heparinases; Heparin hexasaccharide fragment;HMV833; Human chorionic gonadotropin (hCG); IM-862; Interferonalpha/beta/gamma; Interferon inducible protein (IP-10); Interleukin-12;Kringle 5 (plasminogen fragment); Marimastat; Metalloproteinaseinhibitors (TIMPs); 2-Methoxyestradiol; MMI 270 (CGS 27023A); MoAbIMC-1C11; Neovastat; NM-3; Panzem; PI-88; Placental ribonucleaseinhibitor; Plasminogen activator inhibitor; Platelet factor-4 (PF4);Prinomastat; Prolactin 16 kD fragment; Proliferin-related protein (PRP);PTK 787/ZK 222594; Retinoids; Solimastat; Squalamine; SS 3304; SU 5416;SU6668; SU11248; Tetrahydrocortisol-S; tetrathiomolybdate; thalidomide;Thrombospondin-1 (TSP-1); TNP-470; Transforming growth factor-beta(TGF-b); Vasculostatin; Vasostatin (calreticulin fragment); ZD6126; ZD6474; farnesyl transferase inhibitors (FTI); and bisphosphonates.

Anti-cancer agents that can be used in combination with the molecules ofthe invention in the various embodiments of the invention, includingpharmaceutical compositions and dosage forms and kits of the invention,include, but are not limited to: acivicin; aclarubicin; acodazolehydrochloride; acronine; adozelesin; aldesleukin; altretamine;ambomycin; ametantrone acetate; aminoglutethimide; amsacrine;anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa;azotomycin; batimastat; benzodepa; bicalutamide; bisantrenehydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate;brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone;caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine;dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;estramustine; estramustine phosphate sodium; etanidazole; etoposide;etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine;fenretinide; floxuridine; fludarabine phosphate; fluorouracil;fluorocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabinehydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;ilmofosine; interleukin II (including recombinant interleukin II, orrIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-n1;interferon alfa-n3; interferon beta-I a; interferon gamma-I b;iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole;leuprolide acetate; liarozole hydrochloride; lometrexol sodium;lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride. Other anti-cancer drugs include,but are not limited to: 20-epi-1,25 dihydroxyvitamin D3;5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein-1; antiandrogen, prostatic carcinoma; antiestrogen;antineoplaston; antisense oligonucleotides; aphidicolin glycinate;apoptosis gene modulators; apoptosis regulators; apurinic acid;ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel;docosanol; dolasetron; doxifluridine; droloxifene; dronabinol;duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab;eflornithine; elemene; emitefur; epirubicin; epristeride; estramustineanalogue; estrogen agonists; estrogen antagonists; etanidazole;etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide;filgrastim; finasteride; flavopiridol; flezelastine; fluasterone;fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane;fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathioneinhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin;ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine;ilomastat; imidazoacridones; imiquimod; immunostimulant peptides;insulin-like growth factor-1 receptor inhibitor; interferon agonists;interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-;iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sd±1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofuran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer. Preferred additional anti-cancer drugs are 5-fluorouraciland leucovorin.

Examples of therapeutic antibodies that can be used in methods of theinvention include but are not limited to ZENAPAX® (daclizumab) anti-CD25antibody (Roche Pharmaceuticals, Switzerland) which is animmunosuppressive, humanized anti-CD25 monoclonal antibody for theprevention of acute renal allograft rejection; PANOREX™ anti-17-IAantibody which is a murine anti-17-IA cell surface antigen IgG2aantibody (Glaxo Wellcome/Centocor); BEC2™ anti-idiotype (GD3 epitope)antibody which is a murine anti-idiotype (GD3 epitope) IgG antibody(ImClone System); IMC-C225™ anti-EGFR antibody which is a chimericanti-EGFR IgG antibody (ImClone System); VITAXIN™ anti-αVβ3 antibodywhich is a humanized anti-αVβ3 integrin antibody (Applied MolecularEvolution/MedImmune); Smart M195™ anti-CD33 antibody which is ahumanized anti-CD33 IgG antibody (Protein Design Lab/Kanebo);LYMPHOCIDE™ ™ anti-CD22 antibody which is a humanized anti-CD22 IgGantibody (Immunomedics); ICM3™ anti-ICAM3 antibody is a humanizedanti-ICAM3 antibody (ICOS Pharm); IDEC-114™ anti-CD80 antibody is aprimatied primatized anti-CD80 antibody (IDEC Pharm/Mitsubishi);IDEC-131™ anti-CD40L antibody is a humanized anti-CD40L antibody(IDEC/Eisai); IDEC-151™ anti-CD4 antibody is a primatized anti-CD4antibody (IDEC); IDEC-152™ anti-CD23 antibody is a primatized anti-CD23antibody (IDEC/Seikagaku); SMART anti-CD3™ anti-CD3 antibody is ahumanized anti-CD3 IgG (Protein Design Lab); 5G1.1™ anti-CS antibody isa humanized anti-complement factor 5 (C5) antibody (Alexion Pharm);D2E7™ anti-TNF-α antibody is a humanized anti-TNF-α antibody (CAT/BASF);CDP870™ anti-TNFα antibody is a humanized anti-TNF-α Fab fragment(Celltech); IDEC-151™ anti-CD4 antibody is a primatized anti-CD4 IgG1antibody (IDEC Pharm/SmithKline Beecham); MDX-CD4™ anti-CD4 antibody isa human anti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571™ anti-TNF-αantibody is a humanized anti-TNF-α IgG4 antibody (Celltech); LDP-02™anti-α4β7 antibody is a humanized anti-α4β7 antibody(LeukoSite/Genentech); OrthoClone OKT4A® anti-CD4 antibody is ahumanized anti-CD4 IgG antibody (Ortho Biotech); ANTOVA™ anti-CD40Lantibody is a humanized anti-CD40L IgG antibody (Biogen); ANTEGREN™anti-VLA-4 antibody is a humanized anti-VLA-4 IgG antibody (Elan); andCAT-152™ anti-TGF-β₂ antibody is a human anti-TGF-β₂ antibody (CambridgeAb Tech). Other examples of therapeutic antibodies that can be used inaccordance with the invention are presented in Table 11.

5.4.2 Autoimmune Disease and Inflammatory Diseases

In some embodiments, molecules of the invention comprise a variant Fcregion, having one or more amino acid modifications in one or moreregions, which modification increases the affinity of the variant Fcregion for FcγRIIB but decreases the affinity of the variant Fc regionfor FcγRIIIA and/or FcγRIIA. Molecules of the invention with suchbinding characteristics are useful in regulating the immune response,e.g., in inhibiting the immune response in connection with autoimmunediseases or inflammatory diseases. Although not intending to be bound byany mechanism of action, molecules of the invention with an enhancedaffinity for FcγRIIB and a decreased affinity for FcγRIIIA and/orFcγRIIA may lead to dampening of the activating response to FcγR andinhibition of cellular responsiveness.

In some embodiments, a molecule of the invention comprising a variant Fcregion is not an immunoglobulin, and comprises at least one amino acidmodification which modification increases the affinity of the variant Fcregion for FcγRIIB relative to a molecule comprising a wild-type Fcregion. In other embodiments, said molecule further comprises one ormore amino acid modifications, which modifications decreases theaffinity of the molecule for an activating FcγR. In some embodiments,the molecule is a soluble (i.e., not membrane bound) Fc region. Theinvention contemplates other amino acid modifications within the solubleFc region which modulate its affinity for various Fc receptors,including those known to one skilled in the art as described herein. Inother embodiments, the molecule (e.g., the Fc region comprising at leastone or more amino acid modification) is modified using techniques knownto one skilled in the art and as described herein to increase the invivo half life of the Fc region. Such molecules have therapeutic utilityin treating and/or preventing an autoimmune disorder. Although notintending to be bound by any mechanism of actions, such molecules withenhanced affinity for FcγRIIB will lead to a dampening of the activatingreceptors and thus a dampening of the immune response and havetherapeutic efficacy for treating and/or preventing an autoimmunedisorder.

In certain embodiments, the one or more amino acid modifications, whichincrease the affinity of the variant Fc region for FcγRIIB but decreasethe affinity of the variant Fc region for FcγRIIIA comprise asubstitution at position 246 with threonine and at position 396 withhistidine; or a substitution at position 268 with aspartic acid and atposition 318 with aspartic acid; or a substitution at position 217 withserine, at position 378 with valine, and at position 408 with arginine;or a substitution at position 375 with cysteine and at position 396 withleucine; or a substitution at position 246 with isoleucine and atposition 334 with asparagine. In one embodiment, the one or more aminoacid modifications, which increase the affinity of the variant Fc regionfor FcγRIIB but decrease the affinity of the variant Fc region forFcγRIIIA comprise a substitution at position 247 with leucine. Inanother embodiment, the one or more amino acid modification, whichincreases the affinity of the variant Fc region for FcγRIIB butdecreases the affinity of the variant Fc region for FcγRIIIA comprise asubstitution at position 372 with tyrosine. In yet another embodiment,the one or more amino acid modification, which increases the affinity ofthe variant Fc region for FcγRIIB but decreases the affinity of thevariant Fc region for FcγRIIIA comprise a substitution at position 326with glutamic acid. In one embodiment, the one or more amino acidmodification, which increases the affinity of the variant Fc region forFcγRIIB but decreases the affinity of the variant Fc region for FcγRIIIAcomprise a substitution at position 224 with leucine.

The variant Fc regions that have an enhanced affinity for FcγRIIB and adecreased affinity for FcγRIIIA and/or FcγRIIA relative to a comparablemolecule comprising a wild-type Fc region, may be used to treat orprevent autoimmune diseases or inflammatory diseases. The presentinvention provides methods of preventing, treating, or managing one ormore symptoms associated with an autoimmune or inflammatory disorder ina subject, comprising administering to said subject a therapeutically orprophylactically effective amount of one or more molecules of theinvention with variant Fc regions that have an enhanced affinity forFcγRIIB and a decreased affinity for FcγRIIIA and or FcγRIIA relative toa comparable molecule comprising a wild type Fc region.

The invention also provides methods for preventing, treating, ormanaging one or more symptoms associated with an inflammatory disorderin a subject further comprising, administering to said subject atherapeutically or prophylactically effective amount of one or moreanti-inflammatory agents. The invention also provides methods forpreventing, treating, or managing one or more symptoms associated withan autoimmune disease further comprising, administering to said subjecta therapeutically or prophylactically effective amount of one or moreimmunomodulatory agents. Section 5.4.2.1 provides non-limiting examplesof anti-inflammatory agents and immunomodulatory agents.

Examples of autoimmune disorders that may be treated by administeringthe molecules of the present invention include, but are not limited to,alopecia greata, ankylosing spondylitis, antiphospholipid syndrome,autoimmune Addison's disease, autoimmune diseases of the adrenal gland,autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritisand orchitis, autoimmune thrombocytopenia, Behcet's disease, bullouspemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigueimmune dysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CRESTsyndrome, cold agglutinin disease, Crohn's disease, discoid lupus,essential mixed cryoglobulinemia, fibromyalgia-fibromyositis,glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto'sthyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopeniapurpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupuserthematosus, Ménière's disease, mixed connective tissue disease,multiple sclerosis, type 1 or immune-mediated diabetes mellitus,myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritisnodosa, polychrondritis, polyglandular syndromes, polymyalgiarheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoidarthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-mansyndrome, systemic lupus erythematosus, lupus erythematosus, takayasuarteritis, temporal arteristis/giant cell arteritis, ulcerative colitis,uveitis, vasculitides such as dermatitis herpetiformis vasculitis,vitiligo, and Wegener's granulomatosis. Examples of inflammatorydisorders include, but are not limited to, asthma, encephilitis,inflammatory bowel disease, chronic obstructive pulmonary disease(COPD), allergic disorders, septic shock, pulmonary fibrosis,undifferentitated spondyloarthropathy, undifferentiated arthropathy,arthritis, inflammatory osteolysis, and chronic inflammation resultingfrom chronic viral or bacteria infections. Examples of inflammatorydisorders which can be prevented, treated or managed in accordance withthe methods of the invention include, but are not limited to, asthma,encephilitis, inflammatory bowel disease, chronic obstructive pulmonarydisease (COPD), allergic disorders, septic shock, pulmonary fibrosis,undifferentitated spondyloarthropathy, undifferentiated arthropathy,arthritis, inflammatory osteolysis, and chronic inflammation resultingfrom chronic viral or bacteria infections.

Molecules of the invention with variant Fc regions that have an enhancedaffinity for FcγRIIB and a decreased affinity for FcγRIIIA relative to acomparable molecule comprising a wild-type Fc region can also be used toreduce the inflammation experienced by animals, particularly mammals,with inflammatory disorders. In a specific embodiment, a molecule of theinvention reduces the inflammation in an animal by at least 99%, atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 60%, at least 50%, at least 45%, at least 40%, atleast 45%, at least 35%, at least 30%, at least 25%, at least 20%, or atleast 10% relative to the inflammation in an animal, which is notadministered the said molecule or which is administered the parentmolecule.

Molecules of the invention with variant Fc regions that have an enhancedaffinity for FcγRIIB and a decreased affinity for FcγRIIIA relative to acomparable molecule comprising a wild-type Fc region can also be used toprevent the rejection of transplants.

The invention further contemplates engineering any of the antibodiesknown in the art for the treatment and/or prevention of autoimmunedisease or inflammatory disease, so that the antibodies comprise avariant Fc region comprising one or more amino acid modifications, whichhave been identified by the methods of the invention to have a conferredeffector function and/or enhanced affinity for FcγRIIB and a decreasedaffinity for FcγRIIIA relative to a comparable molecule comprising awild type Fc region. A non-limiting example of the antibodies that areused for the treatment or prevention of inflammatory disorders which canbe engineered according to the invention is presented in Table 12A, anda non-limiting example of the antibodies that are used for the treatmentor prevention of autoimmune disorder is presented in Table 12B.

TABLE 12A ANTIBODIES FOR INFLAMMATORY DISEASES AND AUTOIMMUNE DISEASESTHAT CAN ENGINEERED IN ACCORDANCE WITH THE INVENTION. Antibody TargetProduct Name Antigen Type Isotype Sponsors Indication 5G1.1 ComplementHumanized IgG Alexion Rheumatoid (C5) Pharm Inc Arthritis 5G1.1Complement Humanized IgG Alexion SLE (C5) Pharm Inc 5G1.1 ComplementHumanized IgG Alexion Nephritis (C5) Pharm Inc 5G1.1-SC ComplementHumanized ScFv Alexion Cardiopulmonary (C5) Pharm Inc Bypass 5G1.1-SCComplement Humanized ScFv Alexion Myocardial (C5) Pharm Inc Infarction5G1.1-SC Complement Humanized ScFv Alexion Angioplasty (C5) Pharm IncABX-CBL CBL Human Abgenix Inc GvHD ABX-CBL CD147 Murine IgG Abgenix IncAllograft rejection ABX-IL8 IL-8 Human IgG2 Abgenix Inc PsoriasisAntegren VLA-4 Humanized IgG Athena/Elan Multiple Sclerosis Anti- CD11aHumanized IgG1 Genentech Psoriasis CD11a Inc/Xoma Anti- CD18 HumanizedFab′2 Genentech Inc Myocardial CD18 infarction Anti- CD18 Murine Fab′2Pasteur- Allograft rejection LFA1 Merieux/ Immunotech Antova CD40LHumanized IgG Biogen Allograft rejection Antova CD40L Humanized IgGBiogen SLE BTI-322 CD2 Rat IgG Medimmune GvHD, Psoriasis Inc CDP571TNF-alpha Humanized IgG4 Celltech Crohn's CDP571 TNF-alpha HumanizedIgG4 Celltech Rheumatoid Arthritis CDP850 E-selectin Humanized CelltechPsoriasis Corsevin M Fact VII Chimeric Centocor Anticoagulant D2E7TNF-alpha Human CAT/BASF Rheumatoid Arthritis Hu23F2G CD11/18 HumanizedICOS Pharm Multiple Sclerosis Inc Hu23F2G CD11/18 Humanized IgG ICOSPharm Stroke Inc IC14 CD14 ICOS Pharm Toxic shock Inc ICM3 ICAM-3Humanized ICOS Pharm Psoriasis Inc IDEC-114 CD80 Primatised IDECPsoriasis Pharm/Mitsubishi IDEC-131 CD40L Humanized IDEC SLE Pharm/EisaiIDEC-131 CD40L Humanized IDEC Multiple Sclerosis Pharm/Eisai IDEC-151CD4 Primatised IgG1 IDEC Rheumatoid Pharm/Glaxo Arthritis SmithKlineIDEC-152 CD23 Primatised IDEC Pharm Asthma/Allergy Infliximab TNF-alphaChimeric IgG1 Centocor Rheumatoid Arthritis Infliximab TNF-alphaChimeric IgG1 Centocor Crohn's LDP-01 beta2- Humanized IgG MillenniumStroke integrin Inc (LeukoSite Inc.) LDP-01 beta2- Humanized IgGMillennium Allograft rejection integrin Inc (LeukoSite Inc.) LDP-02alpha4beta7 Humanized Millennium Ulcerative Colitis Inc (LeukoSite Inc.)MAK- TNF alpha Murine Fab′2 Knoll Pharm, Toxic shock 195F BASF MDX-33CD64 (FcR) Human Medarex/Centeon Autoimmune haematogical disorders MDX-CD4 Human IgG Medarex/Eisai/ Rheumatoid CD4 Genmab Arthritis MEDI-507CD2 Humanized Medimmune Psoriasis Inc MEDI-507 CD2 Humanized MedimmuneGvHD Inc OKT4A CD4 Humanized IgG Ortho Biotech Allograft rejectionOrthoClone CD4 Humanized IgG Ortho Biotech Autoimmune OKT4A diseaseOrthoclone/ CD3 Murine mIgG2a Ortho Biotech Allograft rejection anti-CD3OKT3 RepPro/ gpIIbIIIa Chimeric Fab Centocor/Lilly Complications ofAbciximab coronary angioplasty rhuMab- IgE Humanized IgG1Genentech/Novartis/ Asthma/Allergy E25 Tanox Biosystems SB-240563 IL5Humanized GlaxoSmithKline Asthma/Allergy SB-240683 IL-4 HumanizedGlaxoSmithKline Asthma/Allergy SCH55700 IL-5 Humanized Celltech/ScheringAsthma/Allergy Simulect CD25 Chimeric IgG1 Novartis Allograft rejectionPharm SMART CD3 Humanized Protein Autoimmune a-CD3 Design Lab diseaseSMART CD3 Humanized Protein Allograft rejection a-CD3 Design Lab SMARTCD3 Humanized IgG Protein Psoriasis a-CD3 Design Lab Zenapax CD25Humanized IgG1 Protein Allograft rejection Design Lab/Hoffman- La Roche

TABLE 12B ANTIBODIES FOR AUTOIMMUNE DISORDERS THAT CAN BE ENGINEERED INACCORDANCE WITH THE INVENTION Antibody Indication Target Antigen ABX-RB2antibody to CBL antigen on T cells, B cells and NK cells fully humanantibody from the Xenomouse 5c8 (Anti CD-40 Phase II trials were haltedin October CD-40 ligand antibody) 1999 examine “adverse events” IDEC 131systemic lupus erythyematous anti CD40 (SLE) humanized IDEC 151rheumatoid arthritis primatized; anti-CD4 IDEC 152 Asthma primatized;anti-CD23 IDEC 114 Psoriasis primatized anti-CD80 MEDI-507 rheumatoidarthritis; multiple anti-CD2 sclerosis Crohn's disease Psoriasis LDP-02(anti-b7 inflammatory bowel disease a4b7 integrin receptor on white mAb)Chron's disease blood cells (leukocytes) ulcerative colitis SMART Anti-autoimmune disorders Anti-Gamma Interferon Gamma Interferon antibodyVerteportin rheumatoid arthritis MDX-33 blood disorders caused bymonoclonal antibody against FcRI autoimmune reactions receptorsIdiopathic Thrombocytopenia Purpurea (ITP) autoimmune hemolytic anemiaMDX-CD4 treat rheumatoid arthritis and other monoclonal antibody againstCD4 autoimmunity receptor molecule VX-497 autoimmune disorders inhibitorof inosine monophosphate multiple sclerosis dehydrogenase rheumatoidarthritis (enzyme needed to make new RNA inflammatory bowel disease andDNA lupus used in production of nucleotides psoriasis needed forlymphocyte proliferation) VX-740 rheumatoid arthritis inhibitor of ICEinterleukin-1 beta (converting enzyme controls pathways leading toaggressive immune response) VX-745 specific to inflammation inhibitor ofP38MAP kinase involved in chemical signalling of mitogen activatedprotein kinase immune response onset and progression of inflammationEnbrel (etanercept) targets TNF (tumor necrosis factor) IL-8 fully humanmonoclonal antibody against IL-8 (interleukin 8) Apogen MP4 recombinantantigen selectively destroys disease associated T-cells inducesapoptosis T-cells eliminated by programmed cell death no longer attackbody's own cells specific apogens target specific T- cells

5.4.2.1 Immunomodulatory Agents and Anti-Inflammatory Agents

The present invention provides methods of treatment for autoimmunediseases and inflammatory diseases comprising administration of themolecules with variant Fc regions having an enhanced affinity forFcγRIIB and a decreased affinity for FcγRIIIA and/or FcγRIIA inconjunction with other treatment agents. Examples of immunomodulatoryagents include, but are not limited to, methothrexate, ENBREL,REMICADE™, leflunomide, cyclophosphamide, cyclosporine A, and macrolideantibiotics (e.g., FK506 (tacrolimus)), methylprednisolone (MP),corticosteroids, steroids, mycophenolate mofetil, rapamycin (sirolimus),mizoribine, deoxyspergualin, brequinar, malononitriloamindes (e.g.,leflunamide), T cell receptor modulators, and cytokine receptormodulators.

Anti-inflammatory agents have exhibited success in treatment ofinflammatory and autoimmune disorders and are now a common and astandard treatment for such disorders. Any anti-inflammatory agentwell-known to one of skill in the art can be used in the methods of theinvention. Non-limiting examples of anti-inflammatory agents includenon-steroidal anti-inflammatory drugs (NSAIDs), steroidalanti-inflammatory drugs, beta-agonists, anticholingeric agents, andmethyl xanthines. Examples of NSAIDs include, but are not limited to,aspirin, ibuprofen, celecoxib (CELEBREX™), diclofenac (VOLTAREN™),etodolac (LODINE™), fenoprofen (NALFON™), indomethacin (INDOCIN™),ketoralac (TORADOL™), oxaprozin (DAYPRO™), nabumentone (RELAFEN™),sulindac (CLINORIL™), tolmentin (TOLECTIN™), rofecoxib (VIOXX™),naproxen (ALEVE™, NAPROSYN™), ketoprofen (ACTRON™) and nabumetone(RELAFEN™). Such NSAIDs function by inhibiting a cyclooxygenase enzyme(e.g., COX-1 and/or COX-2). Examples of steroidal anti-inflammatorydrugs include, but are not limited to, glucocorticoids, dexamethasone(DECADRON™), cortisone, hydrocortisone, prednisone (DELTASONE™),prednisolone, triamcinolone, azulfidine, and eicosanoids such asprostaglandins, thromboxanes, and leukotrienes.

5.4.3 Infectious Disease

The invention also encompasses methods for treating or preventing aninfectious disease in a subject comprising administering atherapeutically or prophylatically effective amount of one or moremolecules of the invention. Infectious diseases that can be treated orprevented by the molecules of the invention are caused by infectiousagents including but not limited to viruses, bacteria, fungi, protozae,and viruses.

Viral diseases that can be treated or prevented using the molecules ofthe invention in conjunction with the methods of the present inventioninclude, but are not limited to, those caused by hepatitis type A,hepatitis type B, hepatitis type C, influenza, varicella, adenovirus,herpes simplex type I (HSV-I), herpes simplex type II (HSV-II),rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytialvirus, papilloma virus, papova virus, cytomegalovirus, echinovirus,arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus,rubella virus, polio virus, small pox, Epstein Barr virus, humanimmunodeficiency virus type I (HIV-I), human immunodeficiency virus typeII (HIV-II), and agents of viral diseases such as viral miningitis,encephalitis, dengue or small pox.

Bacterial diseases that can be treated or prevented using the moleculesof the invention in conjunction with the methods of the presentinvention, that are caused by bacteria include, but are not limited to,mycobacteria rickettsia, mycoplasma, neisseria, S. pneumonia, Borreliaburgdorferi (Lyme disease), Bacillus antracis (anthrax), tetanus,streptococcus, staphylococcus, mycobacterium, tetanus, pertissus,cholera, plague, diptheria, chlamydia, S. aureus and legionella.

Protozoal diseases that can be treated or prevented using the moleculesof the invention in conjunction with the methods of the presentinvention, that are caused by protozoa include, but are not limited to,leishmania, kokzidioa, trypanosoma or malaria.

Parasitic diseases that can be treated or prevented using the moleculesof the invention in conjunction with the methods of the presentinvention, that are caused by parasites include, but are not limited to,chlamydia and rickettsia.

According to one aspect of the invention, molecules of the inventioncomprising variant Fc regions have an enhanced antibody effectorfunction towards an infectious agent, e.g., a pathogenic protein,relative to a comparable molecule comprising a wild-type Fc region.Examples of infectious agents include but are not limited to bacteria(e.g., Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus,Enterococcus faecials, Candida albicans, Proteus vulgaris,Staphylococcus viridans, and Pseudomonas aeruginosa), a pathogen (e.g.,B-lymphotropic papovavirus (LPV); Bordatella pertussis; Borna Diseasevirus (BDV); Bovine coronavirus; Choriomeningitis virus; Dengue virus; avirus, E. coli; Ebola; Echovirus 1; Echovirus-11 (EV); Endotoxin (LPS);Enteric bacteria; Enteric Orphan virus; Enteroviruses; Feline leukemiavirus; Foot and mouth disease virus; Gibbon ape leukemia virus (GALV);Gram-negative bacteria; Heliobacter pylori; Hepatitis B virus (HBV);Herpes Simplex Virus; HIV-1; Human cytomegalovirus; Human coronovirus;Influenza A, B & C; Legionella; Leishmania mexicana; Listeriamonocytogenes; Measles virus; Meningococcus; Morbilliviruses; Mousehepatitis virus; Murine leukemia virus; Murine gamma herpes virus;Murine retrovirus; Murine coronavirus mouse hepatitis virus;Mycobacterium avium-M; Neisseria gonorrhoeae; Newcastle disease virus;Parvovirus B19; Plasmodium falciparum; Pox Virus; Pseudomonas;Rotavirus; Samonella typhiurium; Shigella; Streptococci; T-celllymphotropic virus 1; Vaccinia virus).

In a specific embodiment, molecules of the invention enhance theefficacy of treatment of an infectious disease by enhancing phagocytosisand/or opsonization of the infectious agent causing the infectiousdisease. In another specific embodiment, molecules of the inventionenhance the efficacy of treatment of an infectious disease by enhancingADCC of infected cells causing the infectious disease.

In some embodiments, the molecules of the invention may be administeredin combination with a therapeutically or prophylactically effectiveamount of one or additional therapeutic agents known to those skilled inthe art for the treatment and/or prevention of an infectious disease.The invention contemplates the use of the molecules of the invention incombination with antibiotics known to those skilled in the art for thetreatment and or prevention of an infectious disease. Antibiotics thatcan be used in combination with the molecules of the invention include,but are not limited to, macrolide (e.g., tobramycin (Tobi®)), acephalosporin (e.g., cephalexin (Keflex®), cephradine (Velosef®),cefuroxime (Ceftin®), cefprozil (Cefzil®), cefaclor (Ceclor®), cefixime(Suprax®) or cefadroxil (Duricef®)), a clarithromycin (e.g.,clarithromycin (Biaxin®)), an erythromycin (e.g., erythromycin(EMycin®)), a penicillin (e.g., penicillin V (V-Cillin K® or Pen VeeK®)) or a quinolone (e.g., ofloxacin (Floxin®), ciprofloxacin (Cipro®)or norfloxacin (Noroxin®)), aminoglycoside antibiotics (e.g., apramycin,arbekacin, bambermycins, butirosin, dibekacin, neomycin, neomycin,undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, andspectinomycin), amphenicol antibiotics (e.g., azidamfenicol,chloramphenicol, florfenicol, and thiamphenicol), ansamycin antibiotics(e.g., rifamide and rifampin), carbacephems (e.g., loracarbef),carbapenems (e.g., biapenem and imipenem), cephalosporins (e.g.,cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefozopran,cefpimizole, cefpiramide, and cefpirome), cephamycins (e.g.,cefbuperazone, cefmetazole, and cefminox), monobactams (e.g., aztreonam,carumonam, and tigemonam), oxacephems (e.g., flomoxef, and moxalactam),penicillins (e.g., amdinocillin, amdinocillin pivoxil, amoxicillin,bacampicillin, benzylpenicillinic acid, benzylpenicillin sodium,epicillin, fenbenicillin, floxacillin, penamccillin, penethamatehydriodide, penicillin o-benethamine, penicillin 0, penicillin V,penicillin V benzathine, penicillin V hydrabamine, penimepicycline, andphencihicillin potassium), lincosamides (e.g., clindamycin, andlincomycin), amphomycin, bacitracin, capreomycin, colistin, enduracidin,enviomycin, tetracyclines (e.g., apicycline, chlortetracycline,clomocycline, and demeclocycline), 2,4-diaminopyrimidines (e.g.,brodimoprim), nitrofurans (e.g., furaltadone, and furazolium chloride),quinolones and analogs thereof (e.g., cinoxacin, clinafloxacin,flumequine, and grepagloxacin), sulfonamides (e.g., acetylsulfamethoxypyrazine, benzylsulfamide, noprylsulfamide,phthalylsulfacetamide, sulfachrysoidine, and sulfacytine), sulfones(e.g., diathymosulfone, glucosulfone sodium, and solasulfone),cycloserine, mupirocin and tuberin.

In certain embodiments, the molecules of the invention can beadministered in combination with a therapeutically or prophylacticallyeffective amount of one or more antifungal agents. Antifungal agentsthat can be used in combination with the molecules of the inventioninclude but are not limited to amphotericin B, itraconazole,ketoconazole, fluconazole, intrathecal, flucytosine, miconazole,butoconazole, clotrimazole, nystatin, terconazole, tioconazole,ciclopirox, econazole, haloprogrin, naftifine, terbinafine,undecylenate, and griseofuldin.

In some embodiments, the molecules of the invention can be administeredin combination with a therapeutically or prophylactically effectiveamount of one or more anti-viral agent. Useful anti-viral agents thatcan be used in combination with the molecules of the invention include,but are not limited to, protease inhibitors, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors and nucleoside analogs. Examples of antiviral agents includebut are not limited to zidovudine, acyclovir, gangcyclovir, vidarabine,idoxuridine, trifluridine, and ribavirin, as well as foscarnet,amantadine, rimantadine, saquinavir, indinavir, amprenavir, lopinavir,ritonavir, the alpha-interferons; adefovir, clevadine, entecavir,pleconaril.

5.5 Vaccine Therapy

The invention further encompasses using a composition of the inventionto induce an immune response against an antigenic or immunogenic agent,including but not limited to cancer antigens and infectious diseaseantigens (examples of which are disclosed infra). The vaccinecompositions of the invention comprise one or more antigenic orimmunogenic agents to which an immune response is desired, wherein theone or more antigenic or immunogenic agents is coated with a variantantibody of the invention that has a conferred effector function and/oran enhanced affinity to FcγRIIIA. Although not intending to be bound bya particular mechanism of action, coating an antigenic or immunogenicagent with a variant antibody of the invention that has an enhancedaffinity to FcγRIIIA, enhances the immune response to the desiredantigenic or immunogenic agent by inducing humoral and cell-mediatedresponses. The vaccine compositions of the invention are particularlyeffective in eliciting an immune response, preferably a protectiveimmune response against the antigenic or immunogenic agent.

In some embodiments, the antigenic or immunogenic agent in the vaccinecompositions of the invention comprise a virus against which an immuneresponse is desired. The viruses may be recombinant or chimeric, and arepreferably attenuated. Production of recombinant, chimeric, andattenuated viruses may be performed using standard methods known to oneskilled in the art. The invention encompasses a live recombinant viralvaccine or an inactivated recombinant viral vaccine to be formulated inaccordance with the invention. A live vaccine may be preferred becausemultiplication in the host leads to a prolonged stimulus of similar kindand magnitude to that occurring in natural infections, and therefore,confers substantial, long-lasting immunity. Production of such liverecombinant virus vaccine formulations may be accomplished usingconventional methods involving propagation of the virus in cell cultureor in the allantois of the chick embryo followed by purification.

In a specific embodiment, the recombinant virus is non-pathogenic to thesubject to which it is administered. In this regard, the use ofgenetically engineered viruses for vaccine purposes may require thepresence of attenuation characteristics in these strains. Theintroduction of appropriate mutations (e.g., deletions) into thetemplates used for transfection may provide the novel viruses withattenuation characteristics. For example, specific missense mutationswhich are associated with temperature sensitivity or cold adaption canbe made into deletion mutations. These mutations should be more stablethan the point mutations associated with cold or temperature sensitivemutants and reversion frequencies should be extremely low. RecombinantDNA technologies for engineering recombinant viruses are known in theart and encompassed in the invention. For example, techniques formodifying negative strand RNA viruses are known in the art, see, e.g.,U.S. Pat. No. 5,166,057, which is incorporated herein by reference inits entirety.

Alternatively, chimeric viruses with “suicide” characteristics may beconstructed for use in the intradermal vaccine formulations of theinvention. Such viruses would go through only one or a few rounds ofreplication within the host. When used as a vaccine, the recombinantvirus would go through limited replication cycle(s) and induce asufficient level of immune response but it would not go further in thehuman host and cause disease. Alternatively, inactivated (killed) virusmay be formulated in accordance with the invention. Inactivated vaccineformulations may be prepared using conventional techniques to “kill” thechimeric viruses. Inactivated vaccines are “dead” in the sense thattheir infectivity has been destroyed. Ideally, the infectivity of thevirus is destroyed without affecting its immunogenicity. In order toprepare inactivated vaccines, the chimeric virus may be grown in cellculture or in the allantois of the chick embryo, purified by zonalultracentrifugation, inactivated by formaldehyde or β-propiolactone, andpooled.

In certain embodiments, completely foreign epitopes, including antigensderived from other viral or non-viral pathogens can be engineered intothe virus for use in the intradermal vaccine formulations of theinvention. For example, antigens of non-related viruses such as HIV(gp160, gp120, gp41) parasite antigens (e.g., malaria), bacterial orfungal antigens or tumor antigens can be engineered into the attenuatedstrain.

Virtually any heterologous gene sequence may be constructed into thechimeric viruses of the invention for use in the intradermal vaccineformulations. Preferably, heterologous gene sequences are moieties andpeptides that act as biological response modifiers. Preferably, epitopesthat induce a protective immune response to any of a variety ofpathogens, or antigens that bind neutralizing antibodies may beexpressed by or as part of the chimeric viruses. For example,heterologous gene sequences that can be constructed into the chimericviruses of the invention include, but are not limited to, influenza andparainfluenza hemagglutinin neuraminidase and fusion glycoproteins suchas the FIN and F genes of human PIV3. In yet another embodiment,heterologous gene sequences that can be engineered into the chimericviruses include those that encode proteins with immuno-modulatingactivities. Examples of immuno-modulating proteins include, but are notlimited to, cytokines, interferon type 1, gamma interferon, colonystimulating factors, interleukin-1, -2, -4, -5, -6, -12, and antagonistsof these agents.

In yet other embodiments, the invention encompasses pathogenic cells orviruses, preferably attenuated viruses, which express the variantantibody on their surface.

In alternative embodiments, the vaccine compositions of the inventioncomprise a fusion polypeptide wherein an antigenic or immunogenic agentis operatively linked to a variant antibody of the invention that has anenhanced affinity for FcγRIIIA. Engineering fusion polypeptides for usein the vaccine compositions of the invention is performed using routinerecombinant DNA technology methods and is within the level of ordinaryskill.

The invention further encompasses methods to induce tolerance in asubject by administering a composition of the invention. Preferably acomposition suitable for inducing tolerance in a subject, comprises anantigenic or immunogenic agent coated with a variant antibody of theinvention, wherein the variant antibody has a higher affinity toFcγRIIB. Although not intending to be bound by a particular mechanism ofaction, such compositions are effective in inducing tolerance byactivating the FcγRIIB mediated inhibitory pathway.

5.6 Compositions and Methods of Administering

The invention provides methods and pharmaceutical compositionscomprising molecules of the invention (i.e., antibodies, polypeptides)comprising variant Fc regions. The invention also provides methods oftreatment, prophylaxis, and amelioration of one or more symptomsassociated with a disease, disorder or infection by administering to asubject an effective amount of a fusion protein or a conjugated moleculeof the invention, or a pharmaceutical composition comprising a fusionprotein or a conjugated molecule of the invention. In a preferredaspect, an antibody, a fusion protein, or a conjugated molecule, issubstantially purified (i.e., substantially free from substances thatlimit its effect or produce undesired side-effects). In a specificembodiment, the subject is an animal, preferably a mammal such asnon-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) and aprimate (e.g., monkey such as, a cynomolgous monkey and a human). In apreferred embodiment, the subject is a human. In yet another preferredembodiment, the antibody of the invention is from the same species asthe subject.

Various delivery systems are known and can be used to administer acomposition comprising molecules of the invention (i.e., antibodies,polypeptides), comprising variant Fc regions, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the antibody or fusion protein, receptor-mediated endocytosis(See, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), constructionof a nucleic acid as part of a retroviral or other vector, etc. Methodsof administering a molecule of the invention include, but are notlimited to, parenteral administration (e.g., intradermal, intramuscular,intraperitoneal, intravenous and subcutaneous), epidural, and mucosal(e.g., intranasal and oral routes). In a specific embodiment, themolecules of the invention are administered intramuscularly,intravenously, or subcutaneously. The compositions may be administeredby any convenient route, for example, by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968; 5,985,320;5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078;and PCT Publication Nos. WO 92/19244; WO 97/32572; WO 97/44013; WO98/31346; and WO 99/66903, each of which is incorporated herein byreference in its entirety.

The invention also provides that the molecules of the invention (i.e.,antibodies, polypeptides) comprising variant Fc regions, are packaged ina hermetically sealed container such as an ampoule or sachetteindicating the quantity of antibody. In one embodiment, the molecules ofthe invention are supplied as a dry sterilized lyophilized powder orwater free concentrate in a hermetically sealed container and can bereconstituted, e.g., with water or saline to the appropriateconcentration for administration to a subject. Preferably, the moleculesof the invention are supplied as a dry sterile lyophilized powder in ahermetically sealed container at a unit dosage of at least 5 mg, morepreferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35mg, at least 45 mg, at least 50 mg, or at least 75 mg. The lyophilizedmolecules of the invention should be stored at between 2 and 8° C. intheir original container and the molecules should be administered within12 hours, preferably within 6 hours, within 5 hours, within 3 hours, orwithin 1 hour after being reconstituted. In an alternative embodiment,molecules of the invention are supplied in liquid form in a hermeticallysealed container indicating the quantity and concentration of themolecule, fusion protein, or conjugated molecule. Preferably, the liquidform of the molecules of the invention are supplied in a hermeticallysealed container at least 1 mg/ml, more preferably at least 2.5 mg/ml,at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15mg/kg, at least 25 mg/ml, at least 50 mg/ml, at least 100 mg/ml, atleast 150 mg/ml, at least 200 mg/ml of the molecules.

The amount of the composition of the invention which will be effectivein the treatment, prevention or amelioration of one or more symptomsassociated with a disorder can be determined by standard clinicaltechniques. The precise dose to be employed in the formulation will alsodepend on the route of administration, and the seriousness of thecondition, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

For antibodies encompassed by the invention, the dosage administered toa patient is typically 0.0001 mg/kg to 100 mg/kg of the patient's bodyweight. Preferably, the dosage administered to a patient is between0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kgor 0.01 to 0.10 mg/kg of the patient's body weight. Generally, humanantibodies have a longer half-life within the human body than antibodiesfrom other species due to the immune response to the foreignpolypeptides. Thus, lower dosages of human antibodies and less frequentadministration is often possible. Further, the dosage and frequency ofadministration of antibodies of the invention or fragments thereof maybe reduced by enhancing uptake and tissue penetration of the antibodiesby modifications such as, for example, lipidation.

In one embodiment, the dosage of the molecules of the inventionadministered to a patient are 0.01 mg to 1000 mg/day, when used assingle agent therapy. In another embodiment the molecules of theinvention are used in combination with other therapeutic compositionsand the dosage administered to a patient are lower than when saidmolecules are used as a single agent therapy.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion, by injection, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. Preferably,when administering a molecule of the invention, care must be taken touse materials to which the molecule does not absorb.

In another embodiment, the compositions can be delivered in a vesicle,in particular a liposome (See Langer, Science 249:1527-1533 (1990);Treat et al., in Liposomes in the Therapy of Infectious Disease andCancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365(1989); Lopez-Berestein, ibid., pp. 3 17-327; see generally ibid.).

In yet another embodiment, the compositions can be delivered in acontrolled release or sustained release system. Any technique known toone of skill in the art can be used to produce sustained releaseformulations comprising one or more molecules of the invention. See,e.g., U.S. Pat. No. 4,526,938; PCT publication WO 91/05548; PCTpublication WO 96/20698; Ning et al., 1996, “IntratumoralRadioimmunotheraphy of a Human Colon Cancer Xenograft Using aSustained-Release Gel,” Radiotherapy & Oncology 39:179-189, Song et al.,1995, “Antibody Mediated Lung Targeting of Long-Circulating Emulsions,”PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek etal., 1997, “Biodegradable Polymeric Carriers for a bFGF Antibody forCardiovascular Application,” Pro. Int'l. Symp. Control. Rel. Bioact.Mater. 24:853-854; and Lam et al., 1997, “Microencapsulation ofRecombinant Humanized Monoclonal Antibody for Local Delivery,” Proc.Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which isincorporated herein by reference in its entirety. In one embodiment, apump may be used in a controlled release system (See Langer, supra;Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980,Surgery 88:507; and Saudek et al., 1989, N. Engl. J. Med. 321:574). Inanother embodiment, polymeric materials can be used to achievecontrolled release of antibodies (see e.g., Medical Applications ofControlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.(1974); Controlled Drug Bioavailability, Drug Product Design andPerformance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger andPeppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23:61; See alsoLevy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol.25:351; Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. No.5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat.No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99/15154;and PCT Publication No. WO 99/20253). Examples of polymers used insustained release formulations include, but are not limited to,poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate),poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylicacid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides(PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In yetanother embodiment, a controlled release system can be placed inproximity of the therapeutic target (e.g., the lungs), thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).In another embodiment, polymeric compositions useful as controlledrelease implants are used according to Dunn et al. (See U.S. Pat. No.5,945,155). This particular method is based upon the therapeutic effectof the in situ controlled release of the bioactive material from thepolymer system. The implantation can generally occur anywhere within thebody of the patient in need of therapeutic treatment. In anotherembodiment, a non-polymeric sustained delivery system is used, whereby anon-polymeric implant in the body of the subject is used as a drugdelivery system. Upon implantation in the body, the organic solvent ofthe implant will dissipate, disperse, or leach from the composition intosurrounding tissue fluid, and the non-polymeric material will graduallycoagulate or precipitate to form a solid, microporous matrix (See U.S.Pat. No. 5,888,533).

Controlled release systems are discussed in the review by Langer (1990,Science 249:1527-1533). Any technique known to one of skill in the artcan be used to produce sustained release formulations comprising one ormore therapeutic agents of the invention. See, e.g., U.S. Pat. No.4,526,938; International Publication Nos. WO 91/05548 and WO 96/20698;Ning et al., 1996, Radiotherapy & Oncology 39:179-189; Song et al.,1995, PDA Journal of Pharmaceutical Science & Technology 50:372-397;Cleek et al., 1997, Pro. Int'l. Symp. Control. Rel. Bioact. Mater.24:853-854; and Lam et al., 1997, Proc. Int'l. Symp. Control Rel.Bioact. Mater. 24:759-760, each of which is incorporated herein byreference in its entirety.

In a specific embodiment where the composition of the invention is anucleic acid encoding an antibody, the nucleic acid can be administeredin vivo to promote expression of its encoded antibody, by constructingit as part of an appropriate nucleic acid expression vector andadministering it so that it becomes intracellular, e.g., by use of aretroviral vector (See U.S. Pat. No. 4,980,286), or by direct injection,or by use of microparticle bombardment (e.g., a gene gun; Biolistic,Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (See e.g.,Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc.Alternatively, a nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression by homologousrecombination.

For antibodies, the therapeutically or prophylactically effective dosageadministered to a subject is typically 0.1 mg/kg to 200 mg/kg of thesubject's body weight. Preferably, the dosage administered to a subjectis between 0.1 mg/kg and 20 mg/kg of the subject's body weight and morepreferably the dosage administered to a subject is between 1 mg/kg to 10mg/kg of the subject's body weight. The dosage and frequency ofadministration of antibodies of the invention may be reduced also byenhancing uptake and tissue penetration (e.g., into the lung) of theantibodies or fusion proteins by modifications such as, for example,lipidation.

Treatment of a subject with a therapeutically or prophylacticallyeffective amount of molecules of the invention can include a singletreatment or, preferably, can include a series of treatments. In apreferred example, a subject is treated with molecules of the inventionin the range of between about 0.1 to 30 mg/kg body weight, one time perweek for between about 1 to 10 weeks, preferably between 2 to 8 weeks,more preferably between about 3 to 7 weeks, and even more preferably forabout 4, 5, or 6 weeks. In other embodiments, the pharmaceuticalcompositions of the invention are administered once a day, twice a day,or three times a day. In other embodiments, the pharmaceuticalcompositions are administered once a week, twice a week, once every twoweeks, once a month, once every six weeks, once every two months, twicea year or once per year. It will also be appreciated that the effectivedosage of the molecules used for treatment may increase or decrease overthe course of a particular treatment.

5.6.1 Pharmaceutical Compositions

The compositions of the invention include bulk drug compositions usefulin the manufacture of pharmaceutical compositions (e.g., impure ornon-sterile compositions) and pharmaceutical compositions (i.e.,compositions that are suitable for administration to a subject orpatient) which can be used in the preparation of unit dosage forms. Suchcompositions comprise a prophylactically or therapeutically effectiveamount of a prophylactic and/or therapeutic agent disclosed herein or acombination of those agents and a pharmaceutically acceptable carrier.Preferably, compositions of the invention comprise a prophylactically ortherapeutically effective amount of one or more molecules of theinvention and a pharmaceutically acceptable carrier.

In one particular embodiment, the pharmaceutical composition comprises atherapeutically effective amount of one or more molecules of theinvention comprising a variant Fc region, wherein said variant Fc regionbinds FcγRIIIA and/or FcγRIIA with a greater affinity than a comparablemolecule comprising a wild-type Fc region binds FcγRIIIA and/or FcγRIIAand/or said variant Fc region confers an effector function or mediatesan effector function at least 2-fold more effectively than a comparablemolecule comprising a wild-type Fc region, and a pharmaceuticallyacceptable carrier. In another embodiment, the pharmaceuticalcomposition comprises a therapeutically effective amount of one or moremolecules of the invention comprising a variant Fc region, wherein saidvariant Fc region binds FcγRIIIA with a greater affinity than acomparable molecule comprising a wild-type Fc region binds FcγRIIIA, andsaid variant Fc region binds FcγRIIB with a lower affinity than acomparable molecule comprising a wild-type Fc region binds FcγRIIB,and/or said variant Fc region confers and effector function or mediatesan effector function at least 2-fold more effectively than a comparablemolecule comprising a wild-type Fc region, and a pharmaceuticallyacceptable carrier. In another embodiment, said pharmaceuticalcompositions further comprise one or more anti-cancer agents.

The invention also encompasses pharmaceutical compositions comprising atherapeutic antibody (e.g., tumor specific monoclonal antibody) that isspecific for a particular cancer antigen, comprising one or more aminoacid modifications in the Fc region as determined in accordance with theinstant invention, and a pharmaceutically acceptable carrier.

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant(complete and incomplete), excipient, or vehicle with which thetherapeutic is administered. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Water is a preferred carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like.

Generally, the ingredients of compositions of the invention are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compositions of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include, but are not limited tothose formed with anions such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withcations such as those derived from sodium, potassium, ammonium, calcium,ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

5.6.2 Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingmolecules of the invention, are administered to treat, prevent orameliorate one or more symptoms associated with a disease, disorder, orinfection, by way of gene therapy. Gene therapy refers to therapyperformed by the administration to a subject of an expressed orexpressible nucleic acid. In this embodiment of the invention, thenucleic acids produce their encoded antibody or fusion protein thatmediates a therapeutic or prophylactic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, 1993,Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215. Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, a composition of the invention comprises nucleicacids encoding an antibody, said nucleic acids being part of anexpression vector that expresses the antibody in a suitable host. Inparticular, such nucleic acids have promoters, preferably heterologouspromoters, operably linked to the antibody coding region, said promoterbeing inducible or constitutive, and, optionally, tissue-specific. Inanother particular embodiment, nucleic acid molecules are used in whichthe antibody coding sequences and any other desired sequences areflanked by regions that promote homologous recombination at a desiredsite in the genome, thus providing for intrachromosomal expression ofthe antibody encoding nucleic acids (Koller and Smithies, 1989, Proc.Natl. Acad. Sci. USA 86:8932-8935; and Zijlstra et al., 1989, Nature342:435-438).

In another preferred aspect, a composition of the invention comprisesnucleic acids encoding a fusion protein, said nucleic acids being a partof an expression vector that expresses the fusion protein in a suitablehost. In particular, such nucleic acids have promoters, preferablyheterologous promoters, operably linked to the coding region of a fusionprotein, said promoter being inducible or constitutive, and optionally,tissue-specific. In another particular embodiment, nucleic acidmolecules are used in which the coding sequence of the fusion proteinand any other desired sequences are flanked by regions that promotehomologous recombination at a desired site in the genome, thus providingfor intrachromosomal expression of the fusion protein.

Delivery of the nucleic acids into a subject may be either direct, inwhich case the subject is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the subject. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretroviral or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (See, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432)(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (See, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/20316; WO93/14188; WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, 1989,Proc. Natl. Acad. Sci. USA 86:8932-8935; and Zijlstra et al., 1989,Nature 342:435-438).

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding a molecule of the invention (e.g., an antibody or afusion protein) are used. For example, a retroviral vector can be used(See Miller et al., 1993, Meth. Enzymol. 217:581-599). These retroviralvectors contain the components necessary for the correct packaging ofthe viral genome and integration into the host cell DNA. The nucleicacid sequences encoding the antibody or a fusion protein to be used ingene therapy are cloned into one or more vectors, which facilitatesdelivery of the nucleotide sequence into a subject. More detail aboutretroviral vectors can be found in Boesen et al., (1994, Biotherapy6:291-302), which describes the use of a retroviral vector to deliverthe mdr 1 gene to hematopoietic stem cells in order to make the stemcells more resistant to chemotherapy. Other references illustrating theuse of retroviral vectors in gene therapy are: Clowes et al., 1994, J.Clin. Invest. 93:644-651; Klein et al., 1994, Blood 83:1467-1473;Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossmanand Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson (CurrentOpinion in Genetics and Development 3:499-503, 1993, present a review ofadenovirus-based gene therapy. Bout et al., (Human Gene Therapy, 5:3-10,1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT PublicationWO94/12649; and Wang et al., 1995, Gene Therapy 2:775-783. In apreferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (see, e.g., Walsh et al., 1993, Proc. Soc. Exp. Biol. Med.204:289-300 and U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a subject.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to, transfection, electroporation,microinjection, infection with a viral or bacteriophage vector,containing the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcellmediated gene transfer, spheroplast fusion, etc.Numerous techniques are known in the art for the introduction of foreigngenes into cells (See, e.g., Loeffler and Behr, 1993, Meth. Enzymol.217:599-618, Cohen et al., 1993, Meth. Enzymol. 217:618-644; and Clin.Pharma. Ther. 29:69-92, 1985) and may be used in accordance with thepresent invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a subject by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the subject.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody or a fusion protein areintroduced into the cells such that they are expressible by the cells ortheir progeny, and the recombinant cells are then administered in vivofor therapeutic effect. In a specific embodiment, stem or progenitorcells are used. Any stem and/or progenitor cells which can be isolatedand maintained in vitro can potentially be used in accordance with thisembodiment of the present invention (See e.g., PCT Publication WO94/08598; Stemple and Anderson, 1992, Cell 7 1:973-985; Rheinwald, 1980,Meth. Cell Bio. 21A:229; and Pittelkow and Scott, 1986, Mayo ClinicProc. 61:771).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

5.6.3 Kits

The invention provides a pharmaceutical pack or kit comprising one ormore containers filled with the molecules of the invention (i.e.,antibodies, polypeptides comprising variant Fc regions). Additionally,one or more other prophylactic or therapeutic agents useful for thetreatment of a disease can also be included in the pharmaceutical packor kit. The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises one or more molecules of theinvention. In another embodiment, a kit further comprises one or moreother prophylactic or therapeutic agents useful for the treatment ofcancer or infectious disease, in one or more containers. In anotherembodiment, a kit further comprises one or more cytotoxic antibodiesthat bind one or more antigens associated with cancer or infectiousdisease. In certain embodiments, the other prophylactic or therapeuticagent is a chemotherapeutic. In other embodiments, the prophylactic ortherapeutic agent is a biological or hormonal therapeutic.

5.7 Characterization and Demonstration of Therapeutic Utility

Several aspects of the pharmaceutical compositions, prophylactic, ortherapeutic agents of the invention are preferably tested in vitro, in acell culture system, and in an animal model organism, such as a rodentanimal model system, for the desired therapeutic activity prior to usein humans. For example, assays which can be used to determine whetheradministration of a specific pharmaceutical composition is desired,include cell culture assays in which a patient tissue sample is grown inculture, and exposed to or otherwise contacted with a pharmaceuticalcomposition of the invention, and the effect of such composition uponthe tissue sample is observed. The tissue sample can be obtained bybiopsy from the patient. This test allows the identification of thetherapeutically most effective prophylactic or therapeutic molecule(s)for each individual patient. In various specific embodiments, in vitroassays can be carried out with representative cells of cell typesinvolved in an autoimmune or inflammatory disorder (e.g., T cells), todetermine if a pharmaceutical composition of the invention has a desiredeffect upon such cell types.

Combinations of prophylactic and/or therapeutic agents can be tested insuitable animal model systems prior to use in humans. Such animal modelsystems include, but are not limited to, rats, mice, chicken, cows,monkeys, pigs, dogs, rabbits, etc. Any animal system well-known in theart may be used. In a specific embodiment of the invention, combinationsof prophylactic and/or therapeutic agents are tested in a mouse modelsystem. Such model systems are widely used and well-known to the skilledartisan. Prophylactic and/or therapeutic agents can be administeredrepeatedly. Several aspects of the procedure may vary. Said aspectsinclude the temporal regime of administering the prophylactic and/ortherapeutic agents, and whether such agents are administered separatelyor as an admixture.

Preferred animal models for use in the methods of the invention are, forexample, transgenic mice expressing human FcγRs on mouse effector cells,e.g., any mouse model described in U.S. Pat. No. 5,877,396 (which isincorporated herein by reference in its entirety) can be used in thepresent invention. Transgenic mice for use in the methods of theinvention include, but are not limited to, mice carrying human FcγRIIIA;mice carrying human FcγRIIA; mice carrying human FcγRIIB and humanFcγRIIIA; mice carrying human FcγRIIB and human FcγRIIA.

Preferably, mutations showing the highest levels of activity in thefunctional assays described above will be tested for use in animal modelstudies prior to use in humans. Sufficient quantities of antibodies maybe prepared for use in animal models using methods described supra, forexample, using mammalian expression systems and IgG purification methodsdisclosed and exemplified herein.

Mouse xenograft models may be used for examining efficacy of mouseantibodies generated against a tumor specific target based on theaffinity and specificity of the CDR regions of the antibody molecule andthe ability of the Fc region of the antibody to elicit an immuneresponse (Wu et al., 2001, Trends Cell Biol. 11: S2-9). Transgenic miceexpressing human FcγRs on mouse effector cells are unique and aretailor-made animal models to test the efficacy of human Fc-FcγRinteractions. Pairs of FcγRIIIA, FcγRIIIB and FcγRIIA transgenic mouselines generated in the lab of Dr. Jeffrey Ravetch (Through a licensingagreement with Rockefeller U. and Sloan Kettering Cancer center) can beused such as those listed in the Table 13 below.

TABLE 13 Mice Strains Strain Background Human FcR Nude/CD16A KO noneNude/CD16A KO FcγRIIIA Nude/CD16A KO FcγR IIA Nude/CD16A KO FcγR IIA andIIIA Nude/CD32B KO none Nude/CD32B KO FcγR IIB

Preferably Fc mutants showing both enhanced binding to FcγRIIIA andreduced binding to FcγRIIB, increased activity in ADCC and phagocytosisassays are tested in animal model experiments. The animal modelexperiments examine the increase in efficacy of Fc mutant bearingantibodies in FcγRIIIA transgenic, nude mCD16A knockout mice compared toa control which has been administered native antibody. Preferably,groups of 8-10 mice are examined using a standard protocol. An exemplaryanimal model experiment may comprise the following steps: in a breastcancer model, ˜2×10⁶ SK-BR-3 cells are injected subcutaneously on day 1with 0.1 mL PBS mixed with Matrigel (Becton Dickinson). Initially a wildtype chimeric antibody and isotype control are administered to establisha curve for the predetermined therapeutic dose, intravenous injection of4D5 on day 1 with an initial dose of 4 μg/g followed by weeklyinjections of 2 μg/g. Tumor volume is monitored for 6-8 weeks to measureprogress of the disease. Tumor volume should increase linearly with timein animals injected with the isotype control. In contrast very littletumor growth should occur in the group injected with 4D5. Results fromthe standard dose study are used to set an upper limit for experimentstesting the Fc mutants. These studies are done using subtherapeuticdoses of the Fc mutant containing antibodies. A one tenth dose was usedon xenograft models in experiments done in FcγRIIB knockout mice, see,Clynes et al., 2000, Nat. Med. 6: 443-6, with a resultant block in tumorcell growth. Since the mutants of the invention preferrably show anincrease in FcγRIIIA activation and reduction in FcγRIIB binding themutants are examined at one tenth therapeutic dose. Examination of tumorsize at different intervals indicates the efficacy of the antibodies atthe lower dose. Statistical analysis of the data using t test provides away of determining if the data is significant. Fc mutants that showincreased efficacy are tested at incrementally lower doses to determinethe smallest possible dose as a measure of their efficacy.

The anti-inflammatory activity of the combination therapies of inventioncan be determined by using various experimental animal models ofinflammatory arthritis known in the art and described in Crofford L. J.and Wilder R. L., “Arthritis and Autoimmunity in Animals”, in Arthritisand Allied Conditions: A Textbook of Rheumatology, McCarty et al.(eds.), Chapter 30 (Lee and Febiger, 1993). Experimental and spontaneousanimal models of inflammatory arthritis and autoimmune rheumaticdiseases can also be used to assess the anti-inflammatory activity ofthe combination therapies of invention. The following are some assaysprovided as examples, and not by limitation.

The principle animal models for arthritis or inflammatory disease knownin the art and widely used include: adjuvant-induced arthritis ratmodels, collagen-induced arthritis rat and mouse models andantigen-induced arthritis rat, rabbit and hamster models, all describedin Crofford L. J. and Wilder R. L., “Arthritis and Autoimmunity inAnimals”, in Arthritis and Allied Conditions: A Textbook ofRheumatology, McCarty et al.(eds.), Chapter 30 (Lee and Febiger, 1993),incorporated herein by reference in its entirety.

The anti-inflammatory activity of the combination therapies of inventioncan be assessed using a carrageenan-induced arthritis rat model.Carrageenan-induced arthritis has also been used in rabbit, dog and pigin studies of chronic arthritis or inflammation. Quantitativehistomorphometric assessment is used to determine therapeutic efficacy.The methods for using such a carrageenan-induced arthritis model isdescribed in Hansra P. et al., “Carrageenan-Induced Arthritis in theRat,” Inflammation, 24(2): 141-155, (2000). Also commonly used arezymosan-induced inflammation animal models as known and described in theart.

The anti-inflammatory activity of the combination therapies of inventioncan also be assessed by measuring the inhibition of carrageenan-inducedpaw edema in the rat, using a modification of the method described inWinter C. A. et al., “Carrageenan-Induced Edema in Hind Paw of the Ratas an Assay for Anti-inflammatory Drugs” Proc. Soc. Exp. Biol Med. 111,544-547, (1962). This assay has been used as a primary in vivo screenfor the anti-inflammatory activity of most NSAIDs, and is consideredpredictive of human efficacy. The anti-inflammatory activity of the testprophylactic or therapeutic agents is expressed as the percentinhibition of the increase in hind paw weight of the test group relativeto the vehicle dosed control group.

Animal models for autoimmune disorders can also be used to assess theefficacy of the combination therapies of invention. Animal models forautoimmune disorders such as type 1 diabetes, thyroid autoimmunity,systemic lupus eruthematosus, and glomerulonephritis have been developed(Flanders et al., 1999, Autoimmunity 29:235-246; Krogh et al., 1999,Biochimie 81:511-515; Foster, 1999, Semin. Nephrol. 19:12-24).

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of thecombinatorial therapies disclosed herein for autoimmune and/orinflammatory diseases.

Toxicity and efficacy of the prophylactic and/or therapeutic protocolsof the instant invention can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Prophylacticand/or therapeutic agents that exhibit large therapeutic indices arepreferred. While prophylactic and/or therapeutic agents that exhibittoxic side effects may be used, care should be taken to design adelivery system that targets such agents to the site of affected tissuein order to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the prophylactic and/ortherapeutic agents for use in humans. The dosage of such agents liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any agent used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

The anti-cancer activity of the therapies used in accordance with thepresent invention also can be determined by using various experimentalanimal models for the study of cancer such as the SCID mouse model ortransgenic mice or nude mice with human xenografts, animal models, suchas hamsters, rabbits, etc. known in the art and described in Relevanceof Tumor Models for Anticancer Drug Development (1999, eds. Fiebig andBurger); Contributions to Oncology (1999, Karger); The Nude Mouse inOncology Research (1991, eds. Boven and Winograd); and Anticancer DrugDevelopment Guide (1997 ed. Teicher), herein incorporated by referencein their entireties.

Preferred animal models for determining the therapeutic efficacy of themolecules of the invention are mouse xenograft models. Tumor cell linesthat can be used as a source for xenograft tumors include but are notlimited to, SKBR3 and MCF7 cells, which can be derived from patientswith breast adenocarcinoma. These cells have both erbB2 and prolactinreceptors. SKBR3 cells have been used routinely in the art as ADCC andxenograft tumor models. Alternatively, OVCAR3 cells derived from a humanovarian adenocarcinoma can be used as a source for xenograft tumors.

The protocols and compositions of the invention are preferably tested invitro, and then in vivo, for the desired therapeutic or prophylacticactivity, prior to use in humans. Therapeutic agents and methods may bescreened using cells of a tumor or malignant cell line. Many assaysstandard in the art can be used to assess such survival and/or growth;for example, cell proliferation can be assayed by measuring ³H-thymidineincorporation, by direct cell count, by detecting changes intranscriptional activity of known genes such as proto-oncogenes (e.g.,fos, myc) or cell cycle markers; cell viability can be assessed bytrypan blue staining, differentiation can be assessed visually based onchanges in morphology, decreased growth and/or colony formation in softagar or tubular network formation in three-dimensional basement membraneor extracellular matrix preparation, etc.

Compounds for use in therapy can be tested in suitable animal modelsystems prior to testing in humans, including but not limited to inrats, mice, chicken, cows, monkeys, rabbits, hamsters, etc., forexample, the animal models described above. The compounds can then beused in the appropriate clinical trials.

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of thecombinatorial therapies disclosed herein for treatment or prevention ofcancer, inflammatory disorder, or autoimmune disease.

5.8 Diagnostic Assays

The invention encompasses molecules, e.g., antibodies, with alteredaffinities and avidities for one or more FcγRs. The antibodies of theinvention with enhanced affinity and avidity for one or more FcγRs areparticularly useful in cellular systems (for example for research ordiagnostic purposes) where the FcγRs are expressed at low levels.Although not intending to be bound by a particular mechanism of action,the molecules of the invention with enhanced affinity and avidity for aparticular FcγR are valuable as research and diagnostic tools byenhancing the sensitivity of detection of FcγRs which are normallyundetectable due to a low level of expression.

6. EXAMPLES

Using a yeast display system, mutant human IgG1 heavy chain Fc regionswere screened for modified affinity to different Fc receptors. Inparticular, a mutant Fc library was generated by error prone PCR(Genemorph, Stratagene), and then the mutant Fc proteins were fused tothe Aga2p cell wall protein, which allowed the fusion protein to besecreted extracellularly and displayed on the yeast cell wall.

Soluble forms of the human receptors (FcγRIIIA and FcγRIIB) were cloned.Detection of the IgG1 Fc domains on the yeast cell surface, however, ishindered due to the low affinity of FcγR for its ligand. In order tocircumvent this limitation, soluble FcγR tetrameric complexes wereformed using an AVITAG sequence which could be enzymaticallybiotinylated and subsequently reacted with streptavidin conjugated tophycoerythrin (SA-PE; Molecular Probes) to form soluble tetrameric FcγRcomplexes. ELISA assays confirmed that the soluble FcγR tetramericcomplexes had a higher avidity for human IgG1 relative to the monomericFcγR. Fc fusion proteins on the yeast cell surface also bound thesoluble FcγR tetrameric complexes as assessed by FACS analysis.

The differential binding of the Fc fusion proteins expressed on theyeast cell surface to soluble tetrameric FcγR complexes was monitored bya FACS analysis. Fc fusion proteins with altered affinities for one ormore soluble tetrameric FcγR complexes were thus identified and werethen incorporated into a complete immunoglobulin and expressed inmammalian cells. The mammalian expressed product was used in ELISAassays to confirm the results obtained in the yeast surface displaysystem. Finally, the mutant Fc regions were sequenced to confirm thealtered residue(s).

6.1 Cloning, Expression and Purification of FcγRIIIA

Materials and Methods

Soluble FcγRIIB and FcγRIIIA were cloned as follows. The cDNA clones forthe human FcγR genes (FcγRIIB and FcγRIIIA) were obtained (gift fromRavetch lab). Soluble region of the FcγRIIIA gene (amino acids 7-203)was amplified by PCR (Table 14), digested with BamHI/HindIII and ligatedinto the pET25vector (Novagen). This vector was digested with Sall/Notland a 370 by fragment was gel isolated. The vector hu3A, (gift from J.Ravetch) was digested with BamHI/Sall and a 270 by fragment containingthe N-terminus of FcγRIIIA was isolated. Both fragments were coligatedinto pcDNA3.1 cut with BamH/NotI to create pcDNA3-FcγRIIIA (amino acids1-203). The soluble region of FcγRIIB (amino acids 33-180) was amplifiedby PCR (Table 14), digested with BglII/HindIII and ligated intopET25b(+) (Novagen). This vector was digested with BamHI/NotI and a 140bp fragment was gel isolated. The vector huRIIb1 (gift from J. Ravetch)was digested with BamHI/EcoRI and a 440 bp N-terminal FcγRIIB fragmentwas isolated. Both of these fragments were coligated into pcDNA3.1 cutwith BamHI/Notl to create pcDNA3-FcγRIIB (amino acids 1-180).Recombinant clones were transfected into 293H cells, supernatants werecollected from cell cultures, and soluble recombinant FcγR (rFcγR)proteins were purified on an IgG sepharose column.

Results

Recombinant Soluble FcγRIIIA (rFcγRIIIA) and Recombinant Soluble FcγRIIB(rFcγRIIB) were Purified to Homogeneity

Subsequent to expression and purification of the recombinant solubleFcγR proteins on an IgG sepharose column, the purity and apparentmolecular weight of the recombinant purified soluble receptor proteinswere determined by SDS-PAGE. As shown in FIG. 1, soluble rFcγRIIIA (FIG.2, lane 1) had the expected apparent molecular weight of ˜35 KDa andsoluble rFcγRIIB (FIG. 2, lane 4) had the expected apparent molecularweight of ˜20 KDa. As shown in FIG. 2, soluble rFcγRIIIA migrates as adiffuse “fuzzy” band which has been attributed to the high degree ofglycosylation normally found on FcγRIIIA (Jefferis, et al., 1995 ImmunolLett. 44, 111-117).

6.1.1 Characterization of Purified Recombinant Soluble FcγRIIIA

Materials and Methods

Purified soluble rFcγRIIIA, which was obtained as described above, wasanalyzed for direct binding against human monomeric or aggregated IgGusing an ELISA assay. The plate is coated with 10 ng of solublerFcγRIIIA overnight in 1×PBS. Subsequent to coating, the plate is washedthree times in 1× PBS/0.1% Tween 20. Human IgG, either biotinylatedmonomeric IgG or biotinylated aggregated IgG, is added to the wells at aconcentration ranging from 0.03 mg/mL to 2 mg/mL, and allowed to bind tothe soluble rFcγRIIIA. The reaction is carried out for one hour at 37°C. The plate is washed again three times with 1× PBS/0.1% Tween 20. Thebinding of human IgG to soluble rFcγRIIIA is detected with streptavidinhorseradish peroxidase conjugate by monitoring the absorbance at 650 nm.The absorbance at 650 nm is proportional to the bound aggregated IgG.

In a blocking ELISA experiment, the ability of an FcγRIIIA monoclonalantibody, 3G8, a mouse anti-FcγRIIIA antibody (Pharmingen), to block thebinding of the receptor to aggregated IgG is monitored. The washing andincubation conditions were the same as described above, except thatprior to IgG addition, a 5-fold molar excess of 3G8 was added andallowed to incubate for 30 minutes at 37° C.

Results

Purified, recombinant soluble FcγRIIIA binds aggregated IgG specifically

The direct binding of purified recombinant soluble FcγRIIIA toaggregated and monomeric IgG was tested using an ELISA assay (FIG. 3).At an IgG concentration of 2 μg/ml, strong binding to the aggregated IgGwas observed. However, at a similar concentration, no binding wasdetected to the monomeric IgG. The binding to aggregated IgG was blockedby 3G8, a mouse anti-FcγRIIIA monoclonal antibody that blocks the ligandbinding site, indicating that the aggregated IgG binding is via that ofthe normal FcγRIIIA ligand binding site (FIG. 3). Soluble rFcγRIIB wasalso characterized and shown to bind to IgG with similar characteristicsas the soluble rFcγRIIIA (data not shown).

6.2 Formation of Soluble FcγR Tetrameric Complexes

Materials and Methods

Construction of plasmids for expression of soluble FcRyIIIA and FcRyIIBfused to the AVITAG peptide.

To generate soluble FcγR tetrameric complexes, the soluble region of thehuman FcRgIIIA gene (amino acids 7-203) was amplified by PCR (Table 14),digested with BamHI/HindIII and ligated into the pET25b(+) (Novagen).This vector was digested with SalI/Notl, and a 370 bp fragment wasisolated by agarose gel electrophoresis. The vector hu3A, (gift from J.Ravetch) was digested with BamHI/SalI, and a 270 bp fragment containingthe N-terminus of FcRyIIIA was isolated. Both fragments were coligatedinto pcDNA3.1 (Invitrogen), which had been digested with BamH/NotI tocreate pcDNA3-FcRgIIIA (amino acids 1-203).

The soluble region of FcRyIIB (amino acids 33-180) was amplified by PCR(Table 14), digested with BglII/HindIII and ligated into pET25b(+)(Novagen). This vector was digested with BamHI/NotI, and a 140 bpfragment was isolated by agarose gel electrophoresis. The vector huRIIb₁(gift from J. Ravetch) was digested with BamHI/EcoRI, and a 440 byFcRyIIB N-terminal fragment was isolated. Both of these fragments wereco-ligated into pcDNA3.1, which had been digested with BamHI/Notl tocreate pcDNA3-FcRyIIB (amino acids 1-180). Subsequently, thelinker-AVITAG™ peptide sequence was fused to the C-terminus of bothFcγRIIIA and FcγRIIB. To generate the FcγRIIIA-linker-avitag andFcγRIIB-linker-AVITAG™ peptide constructs, the pcDNA3.1 FcγRIIIA andFcγRIIB constructs were digested with Not I and XbaI (both cut in thevector sequence) and a 86 base pair double stranded oligonucleotideconsisting of NotI site at the 5′ end and XbaI at the 3′ end was ligatedinto the vector. This 86 bp fragment was generated by annealing two 5′phosphorylated reverse complement oligonucleotides (shown in Table 12 as5′ and 3′ linker.avitag primers) with the restrictions sites for NotIand XbaI already pre-designed. Equal volumes of each primer at 100 ngper ul were mixed and the DNA heated to 90° C. for 15 minutes and cooledat room temperature for an hour to anneal. This created adouble-stranded DNA fragment ready to be ligated to thepcDNA3.1-FcγRIIIA and FcγRIIB constructs digested with the respectiveenzymes. Therefore, the pcDNA3.1-FcRyIIIA-linker-AVITAG andpcDNA3.1-FcRyIIB-linker-AVITAG™ peptide, were constructed.

TABLE 14 PRIMERS USED FOR CONSTRUCTION OF FcγR AND IgG VECTORS OligomerSequence 5′ linker.avitag GGCCGCAGGTGGTGGTGGTTCTGGTGGTGGTGGT(SEQ. ID NO. 1) TCTGGTCTGAACGACATCTTCGAGGCTCAGAAAA TCGAATGGCACGAATGAT 3′linker.avitag CTAGATCATTCGTGCCATTCGATTTTCTGAGCCT (SEQ. ID NO. 2)CGAAGATGTCGTTCAGACCAGAACCACCACCACC AGAACCACCACCACCTGC FcRIIIA leftG TTG GAT CCT CCA ACT GCT CTG CTA (SEQ. ID NO. 3) CTT CTA GTT TFcRIIIA right GAA AAG CTT AAA GAA TGA TGA GAT (SEQ. ID NO. 4)GGT TGA CAC T FcRIIBright GAA GTC GAC AAT GAT CCC CAT TGG(SEQ. ID NO. 5) TGA AGA G FcRIIBleft G TTA GAT CTT GCT GTG CTA TTC(SEQ. ID NO. 6) CTG GCT CC IgG1 right ATA GTC GAC CAC TGA TTT ACC CGG(SEQ. ID NO. 7) AGA IgG1left GGAA TTC AAC ACC AAG GTG GAC AAG(SEQ. ID NO. 8) AAA GTT mcr025; ch1 (f′) AAA GGATCC GCG AGC TCA GCC TCC(SEQ. ID NO. 9) ACC AAG G H021 GTCTGCTCGAAGCATTAACC (SEQ. ID NO. 10)

Biotinylation by BirA

Soluble Fc receptors (FcγR) fused to the 15 amino acid AVITAG™ peptidesequence (Avidity, CO) (Schatz P. J., 1993, Biotechology, 11:1138-1143)at the C-terminus of the protein cloned into pcDNA3.1 were generated bytransiently transfecting 293H cells using Lipofectamine 2000 reagent(Invitrogen, CA). Supernatants were collected from the cultures andsoluble FcR proteins were purified by passing the supernatants over anIgG sepharose column. Concentration of the soluble FcR-AVITAG™ peptidefusion protein was quantitated by absorbance at 280 nm. The AVITAG™peptide present on the soluble FcR proteins was biotinylated accordingto the manufacturer's protocol (Avidity, CO) with the E. coli BirAenzyme, a biotin ligase. A 1:100 final dilution of a cocktail ofprotease inhibitors (Sigma catalog #P8849) and 1 mg/ml finalconcentration of Leupeptin (Sigma L-8511) were added to the mixture toprevent degradation of the proteins. The BirA reaction was incubated atroom temperature overnight, following which the solution wasconcentrated using a Biomax 10K-ultrafiltration device (Millipore) bycentrifugation at 3500 rpm at 4° C. The protein was loaded onto an FPLCSuperdex 200 HR 10/30 column (Pharmacia Biotech) in Tris-HCl (20 mM, pH8.0), 50 mM NaCl to separate the labeled soluble FcγR from free biotin.

Determination of the Extent of Biotinylation by Streptavidin Shift Assay

Approximately 80-85% of the protein was biotinylated by the BirA enzyme(Avidity, CO). The streptavidin-shift assay was used to determine theextent of biotinylation of the protein. Biotinylated protein wasincubated with streptavidin (MW 60,000 Daltons) in different ratios.Unbiotinylated protein alone and streptavidin alone are included ascontrols to determine the extent of biotinylation. The incubation iscarried out either on ice for 2 hours or overnight at 4° C. Samples areanalyzed on a 4-12% SDS-PAGE Bis-Tris (Invitrogen, CA) with reducingagent and without boiling of the samples. Streptavidin boundbiotinylated protein migrates as a high molecular weight band. Theextent of biotinylation is estimated by the amount of monomeric proteinleft in the sample. Absence of monomeric low molecular weight speciesand presence of a complex with molecular weight greater thanstreptavidin alone indicates a high degree of biotinylation.

Formation of FcγR Tetrameric Complexes

Formation of FcγR tetrameric complexes was performed according topreviously established methodologies for MHC class I tetramers (SeeBusch, D. H. et al., 1998 Immunity 8:353-362; Altman, J. D. et al.,1996, Science 274: 94-96). The concentration of the biotinylatedmonomeric FcγR was calculated based on absorbance at 280 nm. Onemolecule of streptavidin-phycoerythrin (SA-PE) (Molecular Probes, OR)has the capacity to bind 4 molecules of biotin. A 5:1 molar ratio ofmonomeric biotinylated FcγR to SA-PE (5× monomeric biotinylated FcγR: 1×SA-PE) was used to ensure an excess of biotinylated protein. Thecalculated molecular weight of SA-PE is 300,000 Daltons, therefore 303mL of a 1 mg/mL solution of streptavidin-PE has 1 nmole of SA-PE, whichwas added to 5 nmole of protein. Efficient formation of tetramericprotein requires SA-PE to be added in step-wise increments. Half theamount of SA-PE was added upfront, and the remaining SA-PE was added insmall aliquots every 20-30 minutes at 4° C. in the dark. The intervalsfor the addition of the remaining SA-PE is flexible. After the additionof SA-PE was complete, the solution was concentrated and loaded over anFPLC size exclusion column as above in phosphate buffered saline, at pH7.4. The fraction that eluted in the void volume with a molecular weightgreater than SA-PE alone was collected. Protease inhibitors werereplenished to prevent protein degradation. The solution wasconcentrated and additional protease inhibitors were added to the finalcomplex for storage. The final concentration of the soluble FcγRtetrameric complex was calculated based on the starting concentration ofthe biotinylated monomeric protein. For example, if 500 μg ofbiotinylated protein was used to make the tetrameric complex and thefinal concentrated tetramers were in a volume of 500 μL, theconcentration is estimated to be approximately 1 mg/mL (The lossesincurred during concentration are not taken into account as it isdifficult to accurately determine how much is lost during each step ofthe formation of the tetramers. It is also not possible to take anabsorbance at 280 nm to measure the concentration due to interferencefrom the PE). Soluble FcγR tetrameric complexes were dispensed in smallaliquots at −80° C. for long term storage with protease inhibitors.Sodium azide was not added to these preparations as the tetramers wereused for screening a yeast display library. On thawing an aliquot, thetetramers were stored at 4° C. for up to 1 week.

ELISA Assay for Characterizing the Tetrameric FcγR Complexes

An ELISA was used to characterize the tetrameric FcγR complexes.Maxisorb F96 well plate (Nunc) was coated with 25 ng of human IgG in PBSbuffer, and incubated overnight at 4° C. The plates were washed withPBS/0.5% BSA/0.1% Tween 20 (wash and diluent buffer) before adding thecombination of FcγRIIIA tetramers and test antibodies to determineblocking with 3G8, a mouse anti-human FcγRIIIA antibody as describedbelow: The blocking step was performed as follows: soluble FcγRIIIAtetramers at a fixed 0.5 mg/ml final concentration were pre-incubatedwith antibodies for 1 h at room temperature in buffer, PBS/0.5% BSA/0.1%Tween 20. The final concentrations of the antibodies ranged from 60mg/mL to 0.25 mg/mL. 3G8 is a mouse anti-human FcγRIIIA antibody, andfor the purpose of this experiment, a chimeric version was used, i.e.,the variable region of the antibody is a mouse anti-human FcγRIIIA andthe constant region of the heavy and light chains is from the IgG1 humanregion. A chimeric 4.4.20. D265A was also used in this experiment, whichis an anti-fluorescein antibody, such that the Fc region contains amutation at position 265, where an aspartic acid is substituted withalanine in the human IgG1, which results in a reduced binding to FcγR.This antibody has been characterized previously (See Clynes et al.,2000, Nat. Med. 6: 443-446; Shields et al., 2001, J. Biol. Chem., 276:6591-6604). This antibody was used as negative isotype control.

The antibodies were allowed to bind to FcγRIIIA tetramers, bypre-incubation for 1 hour at room temperature. The mixture was thenadded to the IgG on the washed plate and incubated for and additionalhour at room temperature. The plate was washed with buffer and DJ130c (amouse anti-human FcγRIIIA antibody available from DAKO, Denmark; itsepitope is distinct from that of the 3G8 antibody) at 1:5000 dilutionwas added and allowed to incubate for 1 hr. at room temperature in orderto detect the bound FcγRIIIA tetramers. Unbound antibodies were washedout with buffer and the bound DJ130c was detected with goat anti-mouseperoxidase (Jackson laboratories). This reagent will not detect thehuman Fc. After washing out the unbound peroxidase-conjugated antibody,the substrate, TMB reagent (BioFx), was added to detect the extent ofblocking with 3G8 versus the isotype control and the developed color wasread at 650 nm.

For direct binding of soluble tetrameric FcγRIIIA to IgG by ELISA,maxisorb plates were coated with 25 ng IgG as described above. Thesoluble tetrameric FcγRIIIA were added from 20 mg/mL to 0.1 mg/mL andthe biotinylated monomeric soluble tetrameric FcγRIIIA were added atconcentrations ranging from 20 mg/mL to 0.16 mg/mL. Detection was thesame as above with DJ130c, followed by goat anti-mouse-peroxidaseantibody. Color developed with the TMB reagent and the plate was read at650 nm.

Results

Soluble FcγRIIIA Tetrameric Complex Binds Monomeric Human IgG Via itsNormal Ligand Binding Site

Soluble FcγRIIIA-AVITAG™ peptide fusion proteins were generated,isolated, and analyzed as described in the Material and Methods sectionusing an ELISA assay and were shown to have similar properties as thenon-AVITAG™ peptide soluble FcγRIIIA protein (data not shown). Thefusion proteins were biotinylated, and the tetrameric complexes weregenerated as described above.

The soluble FcγR tetrameric complex was then assessed for binding itsligand, monomeric human IgG, using an ELISA assay. Analysis by ELISAshowed the soluble tetrameric FcγR complexes bind monomeric human IgGspecifically. As shown in FIG. 3A, binding of soluble tetramericFcγRIIIA to monomeric human IgG is blocked by 3G8, a mouse anti-humanFcγIIIA monoclonal antibody, as monitored by the absorbance at 650 nm.On the other hand, the 4-4-20 monoclonal antibody harboring the D265Amutation was not able to block the binding of soluble tetramericFcγRIIIA to monomeric human IgG (FIG. 4A). This experiment thus confirmsthat binding of the soluble tetrameric FcγRIIIA complex occurs throughthe native ligand binding site.

Soluble FcγRIIIA Tetrameric Complex Binds Monomeric Human IgG with aGreater Avidity than Monomeric Soluble FcγRIIIA

The direct binding of soluble tetrameric FcγRIIIA to aggregated humanIgG was assessed using an ELISA assay and compared to the direct bindingof soluble monomeric FcγRIIIA to monometic human IgG. As shown in FIG.4B, soluble tetrameric FcγRIIIA binds human IgG with a higher avidity(8-10 fold) than the soluble monomeric receptor, as monitored by theabsorbance at 450 nm.

The binding of soluble FcγRIIIA tetrameric complex was also assayedusing magnetic beads coated with Fc Fragment purified from IgG1 (FIG.5). Soluble FcγRIIIA tetrameric complex binds to the IgG1 Fc-coatedbeads, under conditions in which monomer binding is not detected.Specificity of binding was shown by pre-incubating the receptor complex,with an anti-FcγRIIIA monoclonal antibody, LNK16, which blocks Fcbinding. This assay further confirms that soluble FcγRIIIA tetramericcomplex binds monomeric IgG through its normal ligand binding site, andthe avidity of the receptor is increased due to multiple binding siteswithin the complex.

6.3 Construction of Yeast Strain for Display of Mutant IgG1 Fc Domains

Materials and Methods

The pYD1 vector (Invitrogen) is derived directly from a yeastreplicating vector, pCT302 (Shusta, et al., 2000 Nat. Biotechnol. 18:754-759, that has been successfully used to display T-cell receptors anda number of scFVs. This plasmid is centromeric and harbors the TRP1 geneenabling a relatively constant copy number of 1-2 plasmids per cell in atrpl yeast strain. Directional cloning into the polylinker places thegene of interest under the control of the GAL1 promoter and in-framewith AGA2. Fusion of the IgG Fc domain to the yeast Aga2p results in theextracellular secretion of the Aga2-Fc fusion protein and subsequentdisplay of the Fc protein on the cell wall via disulfide bonding to theyeast Aga1p protein, which is an integral cell wall protein.

In order to optimize the display levels, different fragments from theIgG1 heavy chain were amplified by PCR and cloned into pYD1.Specifically, the Fc region of the IgG1 heavy chain (allotype IGlm(a);amino acids 206-447) was amplified by PCR (Table 14) from the IMAGEclone 182740, digested with EcoRI/SaII and ligated into the pYD1 vector(Invitrogen). The initial clone from IMAGE contained a deletion of asingle nucleotide at position 319 which was corrected by in vitro sitedirected mutagenesis to construct pYD-GIF206 (Quickchange, Stratagene).

The CH1—CH3 fragment (amino acids 118-447) was amplified from the heavychain clone of the MAb B6.2 in the pCINEO vector using a 5′ oligo(mcr025;chl(f)) and a 3′ oligo (H021) (See Table 14). The fragment wasdigested with BamHI/NotI and ligated into the pYD1 vector to constructpYD-CH1.

FIG. 6 shows a schematic presentation of the constructs. The CH1—CH3construct contains the CH1 domain in addition to the hinge-CH2—CH3domains of the heavy chain, GIF206 contains 6 amino acid residuesupstream of the hinge and GIF227 starts within the hinge region at anendogenous proteolytic cleavage site (Jendeberg et al., 1997J. Immunol.Meth. 201: 25-34).

6.4 Immunolocalization and Characterization of Fc Domains on the YeastCell Wall

Materials and Methods

Constructs containing the Aga2p-Fc fusion proteins and a control vector,pYD1, lacking any insert, were transformed into the yeast strain EBY100(Invitrogen), MATα ura3-52 trpl leu2Δl his3Δ200 pep4::HIS3 prb1Δ1.6Rcan1 GAL::GAL-AGA1, using a standard lithium acetate yeasttransformation protocol (Gietz et al., 1992 Nucleic Acids Res. 20: 1425)Subsequently, tryptophan prototrophs were selected on defined media.Amplification of independent cell populations and induction of Aga1p andthe Aga2p-Fc fusion proteins were accomplished by growth in glucose,followed by growth in media containing galactose as the primary carbonsource for 24-48 hrs at 20° C. Growth in galactose induces expression ofthe Aga2-Fc fusion proteins via the GAL1 promoter, which subsequentlyleads to the display of the Fc fusion proteins on the yeast cellsurface.

Results

FACS Analysis of Fc Fusion Proteins

Expression of Fc fusion proteins on the yeast cell surface was analyzedby immunostaining using a PE-conjugated polyclonal F(ab)₂ goatanti-human FcγR and HP6017 (Sigma) antibody (Jackson ImmununoresearchLaboratories, Inc.). Fluorescence microscopy shows peripheral stainingfor the three Fc fusion proteins. The control strain, harboring vectoralone, shows little or no staining (data not shown). FACS analysis wasused to quantitate the staining (FIG. 7). The yeast strain containingthe CH1—CH3 fusion demonstrated the highest percentage of cells stainedwith both antibodies (FIGS. 7B and F). The GIF227 construct showed thegreatest mean fluorescence intensity (FIG. 7, panels C and G).

Characterization of the Binding of Fc Fusion Proteins Expressed on theYeast cell Surface

The natural context of the Fc and FcγR proteins places the receptor onthe cell surface and the Fc as the soluble ligand; however, the yeast Fcsurface display reverses the geometry of the natural interaction.Detection of the IgG1 Fc proteins on the surface of the yeast cell wallis complicated by both the low affinity of the FcγR for its ligand andthe reverse geometry inherent in the display system. Although the latterpoint cannot be altered, the avidity of the ligand was improved asexplained above by forming soluble FcγR tetrameric complexes, whichallows detection of FcγR binding to the Fc fusion proteins expressed onthe surface yeast cell wall.

To characterize the binding of soluble tetrameric FcγR complexes to thesurface displayed Fc fusion proteins, yeast cells expressing differentFc constructs were incubated with the soluble rFcγRIIIA tetramericcomplex and analyzed by FACS. Yeast cells harboring pYD-CH1, displayingthe wild type CH1—CH3 construct were bound by the soluble rFcγRIIIAtetrameric complex as shown by FACS analysis. The GIF206 and GIF227strains, however, showed little or no binding to the soluble rFcγRIIIAtetrameric complex as shown by FACS analysis (data not shown).

Mutations in the Fc region that block binding to the FcγRs have beenidentified (Shields et al., 2001; J Biol. Chem. 276: 6591-6604). One ofthese mutations, D265A, was incorporated into pYD-CH1 and this mutantwas expressed on the yeast cell surface. These cells were incubated withthe soluble FcγRIIIA tetrameric complex using a high concentration ofligand (0.15 mM of Fc; 7.5 mM of D265A) FACS analysis indicated thatsoluble FcγRIIIA tetrameric complex bound to wild type Fc (FIG. 8A) butsoluble FcγRIIIA tetrameric complex did not bind to the D265A-Fc mutantindicating that FcγR is interacting with the normal FcR binding site inthe lower hinge-CH2 region (FIG. 8B).

Antibodies against the FcγRIIIA ligand binding site blocked binding ofthe soluble FcγRIIIA tetrameric complex to the wild type Fc proteindisplayed on the yeast cell surface wall, as analyzed by FACS (FIG. 9).The binding of soluble FcγRIIIA tetrameric complex was blocked by the3G8 antibody, as well as the LNK16 antibody, another anti-FcγRIIIAmonoclonal antibody (Advanced Immunological) (Tam et al., 1996 J.Immunol. 157:, 1576-1581) and was not blocked by an irrelevant isotypecontrol. Therefore, binding of soluble FcγRIIIA tetrameric complex tothe Fc proteins displayed on the yeast cell surface occurs through thenormal ligand binding site. The limited binding of the FcγRIIIAtetrameric complex indicates that a subpopulation of cells have acorrectly folded Fc that is accessible to FcγR. There are numerousreasons why only a subpopulation of cells may be able to bind theligand, for example, they may be at different stages of cell cycle orthe fusion proteins may not have been exported.

In order to determine the dissociation constant of the FcγRIIIA-tetramerbinding to the Fc fusion proteins on the yeast cell surface, the bindingof a range of FcγRIIIA tetrameric complex was analyzed using FACS.FcγRIIIA tetrameric complex was titrated at concentrations of 1.4 μM to0.0006 μM. Using the mean fluorescence intensity as a measure of bindingaffinity and nonlinear regression analysis, the K_(D) was determined tobe 0.006 μM (+/−0.001) (data not shown).

6.5 Construction of Fc Mutant Library

A mutant Fc library was constructed using primers flanking the Fcfragment in the Fc-CH1 construct and error-prone PCR (Genemorph,Stratagene). The CH1—CH3 insert in vector pYD-CHI was amplified using amutagenic PCR (Genemorph, Stratagene). Five reactions were carried outusing the pYD-upstream and pYD-downstream primers (Invitrogen). Theresultant amplified fragment was digested with XHOI/BamHI and ligatedinto pYD1. The ligation reaction was then transformed into XL10ultracompetent cells (Stratagene), which resulted in ˜1×10⁶transformants, with 80% of the transformants containing inserts.

Sequence analysis of 28 random plasmids from the library indicated amutation frequency ˜2-3 mutations/kb with a breakdown of 40% conservednucleotide changes and 60% of the mutations resulting in amino acidchanges.

The library was transformed into the yeast strain EBY100, MATα ura3-52trp 1 leu2Δ1 his3Δ200 pep4::HIS3 prb1Δ1.6R can 1 GAL GAL-AGA 1::URA3 toa high efficiency, ˜3.3×10⁵ transformants/ug, in 30 independenttransformation reactions to create a total of ˜10⁷ yeast transformants(Gietz, et al., 1992, Nucleic Acids Res. 20: 1425). The library waspooled and amplified by growth in glucose.

6.6 Selection and Analysis of Fc Mutants

Materials and Methods

Elisa Assay for Screening Fc Mutants

ELISA plates (Nunc F96 MaxiSorp Immunoplate) were coated with 50 ml/wellof 0.5 mg/ml BSA-FITC in carbonate buffer at 4° C., and allowed toincubate overnight. Plates were washed with 1×PBS/0.1% Tween 20 (PBST) 3times. 200 ml/well of PBST/0.5% BSA was added and the plates wereincubated for 30 mins at room temperature. Plates were washed threeadditional times with PBST. 50 ml/well of 1:4 diluted 4-4-20 antibody(approximately 3 mg/mL which would lead to a final concentration of0.7-0.8 mg/well) either wild type or containing an Fc mutant, was addedfrom conditional medium in PBST/0.5% BSA and allowed to incubate for 2hrs at room temperature. Plates were washed with PBST three times.Purified, biotinylated monomeric FcγRIIIA at 3 mg/ml (in PBST/0.5% BSA)was added (50 μl/well) to the plates and allowed to incubate for 1.5hours at room temperature. Plates were washed with PBST three times. 50ml/well of a 1:5000 dilution of Streptavidin-HRP(Pharmacia, RPN 123v) inPBST/0.5% BSA was added and the plates were incubated for 30 minutes atroom temperature. Plates were washed with PBST three times. 80 ml/wellof TMB reagent (BioFX) was then added to the plates, and allowed toincubate for 10-15 minutes at room temperature in a dark place. Thereactions were finally stopped by adding 40 ml/well of stop solution(0.18 M sulfuric acid). Plates were then monitored for absorbance at 450nm. After the first screen, the interesting candidates were furtherconfirmed by serial titration of 4-4-20-Fc mutants in the immuno-complexbased binding ELISA. A few modifications were made in this ELISA. Forcoating the plates, 2 mg/ml BSA-FITC was used. Based on IgG quantitationresults, diluted 4-4-20Fc (wild type or mutants) from conditional mediumwas added to a final concentration of 1, 0.5, 0.25, 0.125, 0.063, and 0mg/ml in PBST-/0.5% BSA.

FACS Screen for the Cell Surface Displayed Fc Proteins

Cells were grown in at least 10 mls of HSM-Trp-Ura pH 5.5 with glucosefor 16-24 hrs or until OD₆₀₀ was greater than 2.0. Cells were spun downat ˜2000 rpm for 5 minutes. Cells were resuspended in an equal volume ofHSM-Trp-Ura, pH 7.0 with galactose. In a 125 ml flask, 36 mls ofgalactose media was added, and inoculated with 9 mls of culture, whichwas incubated at 20° C. with shaking for 24-48 hrs. Growth was monitoredby measuring OD₆₀₀ at 8-16 hr intervals. Cells were harvested at 2K rpmfor 5 minutes, and resuspended in an equal volume of 1×PBS, pH 7.4.

Equilibrium Screen:

An appropriate amount of cells was incubated while maintaining an excessof ligand. For example, it is preferred to start with a number of cellsneeded to ensure 10-fold coverage of the library. For the first sortwith a library containing 10⁷ transformants, 10⁸ cells should be used.In fact it is best to start with 10⁹ cells to compensate for loss duringthe staining protocol.

Incubation was typically done in a 1.5 mL tube in volumes of 20-100 mlsfor 1 hour at 4° C. in the dark on a rotator (incubation buffer: 1×PBSpH7.4; 1 mg/ml BSA). Cells were washed once in 500 ml of incubationbuffer and spun down at 4K rpm for 2.5 minutes. Cells were resuspendedin 100 ml incubation buffer and incubated with the second stainingreagent. For Fc-CH1, a F(ab)₂ goat anti-hFc F(ab)₂—FITC antibody(Jackson Immunoresearch Laboratories, Inc.) can be used to stain for CH1expression. Staining was done with 1 mL for 30 minutes. Cells werewashed additionally in 500 mL of incubation buffer and spun down at 4Krpm for 2.5 minutes, resuspended in 1 mL 1×PBS1 mg/mL BSA and analyzedby FACS.

Typical equilibrium screen sort gates and number of cells collected areshown in Table 15.

TABLE 15 SORT GATES AND NUMBER OF CELLS SORTED Sort Gate total cellsscreened cells collected 1^(st)   5% 10⁸ 5 × 10⁶ 2^(nd)   1% 10⁷ 1 × 10⁵3^(rd) 0.2% 10⁷ 2 × 10⁴ 4^(th) 0.2% 10⁷ 2 × 10⁴

After the 3rd and 4th sorts, cells were plated directly on -trp-uraplates to identify individual mutants. This typically recovered ˜200-400colonies per plate. After collection the cells were placed in 10 mLs ofglucose media in a 50 mL conical tube and grown at 30° C. The wholeprocedure was repeated iteratively.

Results

FACS Analysis of Fc Mutants

After induction in galactose media, cells were harvested and co-stainedwith soluble FcγRIIIA tetrameric complex-PE labeled and F(ab₂) of mouseanti-human Fc-FITC labeled (Jackson Immunoresearch Laboratories, Inc.).Cells were analyzed by FACS and sort gates were used to select the cellsthat showed the highest affinity for the soluble FcγRIIIA tetramericcomplex relative to the amount of Fc expression on the cell surface(FIG. 10). For example, a cell containing a mutant Fc that binds betterto the soluble FcγRIIIA tetrameric complex may express fewer Fc fusionproteins on the yeast cell surface, and this cell will be in the lowerleft hand corner of the sort gate.

Four consecutive sorts were done to enrich for those mutants that showedthe highest affinity for the soluble FcγRIIIA tetrameric complex. Thegates for each successive sort were 5.5%, 1%, 0.2% and 0.1%. After thelast sort, cells were plated onto selective media and individualcolonies were isolated. Each individual colony represented a clonalpopulation of cells harboring a single Fc mutant within the Aga2-Fcfusion protein. Initially 32 independent colonies were picked and testedby FACS for binding to soluble FcγRIIIA tetrameric complex (FIG. 11).Eighteen mutants showed an increase in binding intensity as measured bythe percentage of cells bound by soluble FcγRIIIA tetrameric complex andthe mean fluorescence intensity of the bound cells.

Mutations showing an increase in binding to FcγRIIIA were also testedfor binding to soluble FcγRIIB tetrameric complex (FIG. 11). Mostmutations that lead to an increase in binding to the soluble FcγRIIIAtetrameric complex also resulted in detection of FcγRIIB tetramericcomplex staining (FIG. 11). Based on both previous physical and geneticdata, some mutations that increase binding to FcγRIIIA, are expected toalso increase binding to FcγRIIB (Shields et al., 2001, J Biol. Chem.276: 6591-6604; Sondermann et al., 2000, Nature 406: 267-273).

Analysis of Mutants in a 4-4-20 MAb Produced in a Human Cell Line.

Isolation and analysis of mutations in the yeast system allows for fastidentification of novel mutant alleles. The use of a heterologous systemto isolate mutations could result in the identification of mutationsthat enhance binding through an alteration that results in misfolding oralteration in glycosylation that is specific to yeast. To analyze the Fcmutations in an immunoglobulin molecule that is produced in human cells,the mutants were subcloned into a mammalian expression vector,containing the heavy chain of the anti-fluorescein monoclonal antibody,4-4-20 (Kranz et al., 1982 J. Biol. Chem., 257(12): 6987-6995). Themutant 4-4-20 heavy chains were transiently coexpressed with the lightchain clones in the human kidney cell line (293H). Supernatants werecollected and analyzed by ELISA (FIG. 12).

According to the ELISA assay, the majority of the mutants that wereidentified as having an enhanced affinity for the soluble monomericFcγRIIIA complex, in the secondary FACS analysis, also showed anincrease in binding to the soluble FcγRIIIA tetrameric complex whenpresent in the Fc region of the 4-4-20 monoclonal antibody produced inthe human cell line (FIG. 12A). Two mutants, number 16 and number 19,however, showed a decrease in binding to the soluble FcγRIIIA monomericcomplex.

Table 16, summarizes the mutations that have been identified and theircorresponding binding characteristics to FcγRIIIA and FcγRIIB, asdetermined by both yeast display based assays and ELISA. In Table 16,the symbols represent the following: • corresponds to a 1-fold increasein affinity; + corresponds to a 50% increase in affinity; − correspondsto a 1-fold decrease in affinity; → corresponds to no change in affinitycompared to a comparable molecule comprising a wild-type Fc region.

TABLE 16 MUTATIONS IDENTIFIED AND BINDING CHARACTERISTICS Clone IIIA IIB# Mutation sites Domain binding binding 4 A339V, Q347H CH2, CH3 + + 5L251P, S415I CH2, CH3 + + 7 Aga2p-T43I Note: This is a Aga2p- mutationin T43I Aga2P that enhances display. 8 V185M, K218N, R292L, CH1, hinge,CH2, no − D399E CH3 change 12 K290E, L142P CH1, CH2 + not tested 16A141V, H268L, K288E, CH1, CH2 − not tested P291S 19 L133M, P150Y, K205E,CH1, CH2, CH3 − not tested S383N, N384K 21 P396L CH3 • •+ 25 P396H CH3••• •• 6 K392R CH3 no no change change 15 R301C, M252L, S192T CH1, CH2 −not tested 17 N315I CH2 no not tested change 18 S132I CH1 no not testedchange 26 A162V CH1 no not tested change 27 V348M, K334N, F275I, CH1,Ch2 + + Y202M, K147T 29 H310Y, T289A, G337E CH2 − not tested 30 S119F,G371S, Y407N, CH1, CH2, CH3 + no E258D change 31 K409R, S166N CH1, CH3no not tested change 20 S408I, V215I, V125I CH1, hinge, CH3 + no change24 G385E, P247H CH2, CH3 ••• + 16 V379M CH3 •• no change 17 S219Y Hinge• − 18 V282M CH2 • − 31 F275I, K334N, V348M CH2 + no change 35 D401VCH3 + no change 37 V280L, P395S CH2 + − 40 K222N Hinge • no change 41K246T, Y319F CH2 • no change 42 F243I, V379L CH2, CH3 •+ − 43 K334E CH2•+ − 44 K246T, P396H CH2, CH3 • ••+ 45 H268D, E318D CH2 •+ ••••• 49K288N, A330S, P396L CH2, CH3 ••••• ••• 50 F243L, R255L, E318K CH2 • − 53K334E, T359N, T366S CH2, CH3 • no change 54 I377F CH3 •+ + 57 K334I CH2• no change 58 P244H, L358M, V379M, CH2, CH3 •+ •+ N384K, V397M 59K334E, T359N, T366S CH2, CH3 •+ no (independent isolate) change 61 I377F(independent CH3 ••• ••+ isolate) 62 P247L CH2 •• ••+ 64 P217S, A378V,S408R Hinge, CH3 •• ••••+ 65 P247L, I253N, K334N CH2 ••• ••+ 66 K288M,K334E CH2 ••• − 67 K334E, E380D CH2, CH3 •+ − 68 P247L (independentCH2 + •••• isolate) 69 T256S, V305I, K334E, CH2, CH3 •+ no N390S change70 K326E CH2 •+ ••+ 71 F372Y CH3 + •••••+ 72 K326E (independent CH2 + ••isolate) 74 K334E, T359N, T366S CH2, CH3 •• no (independent isolate)change 75 K334E (independent CH2 ••+ no isolate) change 76 P396L(independent CH3 •+ no isolate) change 78 K326E (independent CH2 •• •••+isolate) 79 K246I, K334N CH2 • •••• 80 K334E (independent CH2 • noisolate) change 81 T335N, K370E, A378, CH2, CH3 • no T394M, S424L change82 K320E, K326E CH2 • • 84 H224L Hinge • ••••• 87 S375C, P396L CH3 •+••••+ 89 E233D, K334E CH2 •+ no change 91 K334E (independent CH2 • noisolate) change 92 K334E (independent CH2 • no isolate) change 94 K334E,T359N, T366S, CH2 • no Q386R change

Analysis of soluble FcγRIIB tetrameric complex binding shows that 7 outof the 8 mutants that showed an increase in binding to the solubleFcγRIIIA tetrameric complex also had an increased binding to the solubleFcγRIIB tetrameric complex (FIG. 12B). One mutant, number 8, showed adecrease in binding to the soluble FcγRIIB tetrameric complex. Three ofthe mutants show no difference in binding to either the soluble FcγRIIIAtetrameric complex or the soluble FcγRIIB tetrameric complex, possiblydue to mutations that result in yeast specific alterations.

6.7 ADCC Assay of Fc Mutants

Effector cell preparation: Peripheral blood mononuclear cells (PBMC)were purified by Ficoll-Paque (Pharmacia, 17-1440-02) Ficoll-Paquedensity gradient centrifugation from normal peripheral human blood(Biowhittaker/Poietics, 1 W-406). Blood was shipped the same day atambient temperature, and diluted 1:1 in PBS and glucose (1 g/1 L) andlayered onto Ficoll in 15 mL conical tubes (3 mL Ficoll; 4 mL PBS/blood)or 50 mL conical tubes (15 mL: Ficoll; 20 mL PBS/blood). Centrifugationwas done at 1500 rpm (400 rcf) for 40 minutes at room temperature. ThePBMC layer was removed (approximately 4-6 mL from 50 mL conical tube)and diluted 1:10 in PBS (which contains no Ca²⁺ or Mg²⁺) in a 50 mLconical tube, and spun for an additional ten minutes at 1200 rpm (250rcf) at room temperature. The supernatant was removed and the pelletswere resuspended in 10-12 mL PBS (which contains no Ca²⁺ or Mg²⁺),transferred to 15 mL conical tubes, and spun for another 10 minutes at1200 rpm at room temperature. The supernatant was removed and thepellets were resuspended in a minimum volume (1-2 mL) of media(Isocove's media (IMDM)+10% fetal bovine serum (FBS), 4 mM Gln,Penicillin/Streptomycin (P/S)). The resuspended PBMC were diluted to theappropriate volume for the ADCC assay; two fold dilutions were done inan ELISA 96 well plate (Nunc F96 MaxiSorp Immunoplate). The yield ofPBMC was approximately 3−5×10⁷ cells per 40-50 mL of whole blood.

Target cell preparation: Target cells used in the assay were SK-BR-3(ATCC Accession number HTB-30; Trempe et al., 1976, Cancer Res. 33-41),Raji (ATCC Accession number CCL-86; Epstein et al., 1965, J. Natl.Cancer Inst. 34: 231-40), or Daudi cells (ATCC Accession number CCL-213;Klein et al., 1968, Cancer Res. 28: 1300-10) (resuspended in 0.5 mL IMDMmedia) and they were labeled with europium chelate bis(acetoxymethyl)2,2″:6′,2″ terpyridine 6,6′ dicarboxylate (BATDA reagent; Perkin ElmerDELFIA reagent; C136-100). K562 cells (ATCC Accession number CCL-243)were used as control cells for NK activity. The Daudi and Raji cellswere spun down; the SK-BR-3 cells were trypsinized for 2-5 minutes at37° C., 5% CO₂ and the media was neutralized prior to being spun down at200-350 G. The number of target cells used in the assays was about4−5×10⁶ cells and it did not exceed 5×10⁶ since labeling efficiency wasbest with as few as 2×10⁶ cells. Once the cells were spun down, themedia was aspirated to 0.5 mL in 15 mL Falcon tubes. 2.5 μl of BATDAreagent was added and the mixture was incubated at 37° C., 5% CO₂ for 30minutes. Cells were washed twice in 10 mL PBS and 0.125 mMsulfinpyrazole (“SP”; SIGMA S-9509); and twice in 10 mL assay media(cell media +0.125 mM sulfinpyrazole). Cells were resuspended in 1 mLassay media, counted and diluted.

When SK-BR-3 cells were used as target cells after the first PBS/SPwash, the PBS/SP was aspirated and 500 μg/mL of FITC was added (PIERCE461110) in IMDM media containing SP, Gln, and P/S and incubated for 30minutes at 37° C., 5% CO₂. Cells were washed twice with assay media;resuspended in 1 mL assay media, counted and diluted.

Antibody Opsonization: Once target cells were prepared as describedsupra, they were opsonized with the appropriate antibodies. In the caseof Fc variants, 50 μL of 1×10⁵ cells/mL were added to 2× concentrationof the antibody harboring the Fc variant. Final concentrations were asfollows: Ch-4-4-20 final concentration was 0.5-1 μg/mL; and Ch4D5 finalconcentration was 30 ng/mL-1 ng/mL.

Opsonized target cells were added to effector cells to produce aneffector:target ratio of 75:1 in the case of the 4-4-20 antibodies withFc variants. In the case of the Ch4D5 antibodies with Fc variants,effector:target ratio of 50:1 or 75:1 were achieved. Effective PBMCgradient for the assay ranges from 100:1 to 1:1. Spontaneous release(SR) was measured by adding 100 μL of assay media to the cells; maximalrelease (MR) was measured by adding 4% TX-100. Cells were spun down at200 rpm in a Beckman centrifuge for 1 minute at room temperature at 57G. Cells were incubated for 3-3.5 hours at 37° C., 5% CO₂. Afterincubation, the cells were spun at 1000 rpm in a Beckman centrifuge(about 220×g) for five minutes at 10° C. 20 μl of supernatant wascollected; 2004 of Eu solution was added and the mixture was shaken for15 minutes at room temperature at 120 rpm on a rotary shaker. Thefluorescence was quantitated in a time resolved fluorometer (Victor1420, Perkin Elmer)

Results

As described above, the variant Fc regions were subcloned into amammalian expression vector, containing the heavy chain of theanti-fluoresceine monoclonal antibody, 4-4-20 (Kranz et al., 1982 J.Biol. Chem., 257(12): 6987-6995). The variant 4-4-20 heavy chains weretransiently coexpressed with the light chain clones in the human kidneycell line (293H). Supernatants were collected and analyzed using theADCC assay. FIG. 13 shows that ADCC activity of the mutants isconcentration-dependent. As summarized in Table 8, five immunoglobulinswith variant Fc regions had an enhanced ADCC activity relative to wildtype ch 4-4-20. The five mutants were as follows: MGFc-27 (G316D, A378V,D399E); MGFc-31 (P247L, N421K); MGFc-10 (K288N, A330S, P396L); MGFc-28(N315I, V379M, T394M); MGFc-29 (F243I, V379L, G420V).

Additional 4-4-20 immunoglobulins with variant Fc regions were assayedfor their ADCC activity relative to a 4-4-20 immunoglobulin with awild-type Fc region. These results are summarized in Table 17.

ADCC assays were also carried out using the same protocol as previouslydescribed for the 4-4-20 antibody, however, the variant Fc regions werecloned into a humanized antibody (Ab4D5) which is specific for the humanepidermal growth factor receptor 2 (HER2/neu). In this case, SK-BR-3cells were used as the target cells that were opsonized with a HER2/neuantibody carrying a variant Fc region. HER2/neu is endogenouslyexpressed by the SK-BR-3 cells and therefore present on the surfacethese cells. FIG. 14 shows the ADCC activity of HER2/neu antibodiescarrying variant Fc regions. Table 18 summarizes the results of ADCCactivity of the mutants in the context of the HER2/neu antibody.Normalization was carried out by comparing the concentration of themutant to the wild type antibody required for a specific value ofpercent cell lysis.

As shown in FIG. 14A, MGFc-5 (V379M), MGFc-9 (P243I, V379L), MGFc-10(K288N, A330S, P396L), MGFc-13 (K334E, T359N, T366S), and MGFc-27(G316D, A378V, D399E) mutants that were cloned in to the humanizedanti-HER2/neu antibody exhibited a higher % specific lysis of SK-BR-3cells relative to the wild antibody.

TABLE 17 SUMMARY OF ADCC ACTIVITY OF MUTANTS ADCC Fc Variant 1 ug/ml 0.5ug/ml Label Ref Amino Acid Variation % specific lysis Normalized %specific lysis Normalized MGFc-27 2C4 G316D, A378V, D399E 33% 2.24 22% 3.60 MGFc-31 3B9 P247L, N421K 30% 2.05 17%  2.90 MGFc-10 1E1 K288N,A330S, P396L 24% 1.66 10%  1.67 MGFc-28 2C5 N315I, V379M, T394M 20% 1.3710%  1.69 MGFc-29 3D11 F243I, V379L, G420V 20% 1.35 7% 1.17 ch4-4-20(P54008) 15% 1.00 6% 1.00 MGFc-35 3D2 R255Q, K326E 11% 0.79 3% 0.53MGFc-36 3D3 K218R, G281D, G385R 10% 0.67 5% 0.78 MGFc-30 3A8 F275Y  9%0.64 2% 0.37 MGFc-32 3C8 D280E, S354F, A431D, L441I  9% 0.62 4% 0.75MGFc-33 3C9 K317N, F423deleted  3% 0.18 −1%  −0.22 MGFc-34 3B10 F241L,E258G −1% −0.08 −4%  −0.71 MGFc-26 D265A  1% 0.08 −3%  −0.45

TABLE 18 SUMMARY OF MUTANTS ELISA ELISA 4-4-20 Anti-HER2 Fc FcR3A,FcR2B, IIIA IIB Phagocytosis ADCC ADCC Variant Amino Acid changesK_(D)/Koff K_(D)/K_(off) binding binding (mutant/WT) (mutant/wt)(mutant/wt) Wt none 198/0.170 94/.094 1   1   1 1 1 MGFc 5 V379M160/0.167 70/0.10 2X  N/C 0.86 2.09 1.77 MGFc 9 P243I, V379L 99.7/0.105 120/0.113 1.5X reduced ? 2.25 2.04 MGFc 10 K288N, A330S, P396L 128/0.11533.4/0.050  5X  3X 1.2 2.96 2.50 MGFc 11 F243L, R255L  90/0.07574.7/0.09  1x  reduced 0.8 2.38 1.00 MGFc 13 K334E, T359N, T366S55.20.128 72/0.11 1.5X N/C [ 1.57 3.67 MGFc 14 K288M, K334E 75.4/0.1  95.6/0.089  3X  reduced [ 1.74 MGFc 23 K334E, R292L 70.2/0.105 108/0.107 [ 2.09 1.6 MGFc 27 G316D, A378V, D399E  72/0.117 46/0.06 1.5X14X  1.4 3.60 6.88 MGFc 28 N315I, A379M, D399E 1X  9X 1.37 1.69 1.00MGFc 29 P243I, V379L, G420V 108/0.082 93.4/.101  2.5X 7X 0.93 1.17 1.00MGFc 31 P247L, N421K  62/0.108  66/0.065 3X  N/C 1.35 2.90 1.00 MGFc 37K248M 154/0.175 100/0.091 1.4X reduced 0.98 3.83 0.67 MGFc 38 K392T,P396L  84/0.104  50/0.041 4.5X  2.5X 1.4 3.07 2.50 MGFc 39 E293V, Q295E,A327T 195/0.198  86/0.074 1.4X reduced 1.5 4.29 0.50 MGFc 40 K248M180/0.186 110/0.09  1.4X reduced 1.14 4.03 MGFc 41 H268N, P396L178/0.159 46.6/0.036  2.2X  4.5X 1.96 2.24 0.67 MGFc 43 Y319F, P352L,P396L 125/0.139 55.7/0.041  3.5X 2X 1.58 1.09

6.8 Analysis of Kinetic Parameters of Fc Mutants

Kinetic parameters of the binding of ch4-4-20 antibodies harboring Fcmutants to FcγRIIIA and FcγRIIB were analyzed using a BIAcore assay(BIAcore instrument 1000, BIAcore Inc., Piscataway, N.J.). The FcγRIIIAused in this assay was a soluble monomeric protein, the extracellularregion of FcγRIIIA joined to the linker-AVITAG sequence as described inSection 6.2 supra. The FcγRIIB used in this assay was a soluble dimericprotein prepared in accordance with the methodology described in U.S.Provisional Application No. 60/439,709 filed on Jan. 13, 2003, which isincorporated herein by reference. Briefly, the FcγRIIB used was theextracellular domain of FcγRIIB fused to the hinge-CH2—CH3 domain ofhuman IgG2.

BSA-FITC (36 μg/mL in 10 mM Acetate Buffer at pH 5.0) was immobilized onone of the four flow cells (flow cell 2) of a sensor chip surfacethrough amine coupling chemistry (by modification of carboxymethylgroups with mixture of NHS/EDC) such that about 5000 response units (RU)of BSA-FITC was immobilized on the surface. Following this, theunreacted active esters were “capped off” with an injection of 1MEt-NH₂. Once a suitable surface was prepared, ch 4-4-20 antibodiescarrying the Fc mutations were passed over the surface by one minuteinjections of a 20 μg/mL solution at a 5 μL/mL flow rate. The level ofch-4-4-20 antibodies bound to the surface ranged between 400 and 700 RU.Next, dilution series of the receptor (FcγRIIIA and FcγRIIB-Fc fusionprotein) in HBS-P buffer (10 mM HEPES, 150 mM NaCl, 0.005% SurfactantP20, 3 mM EDTA, pH 7.4) were injected onto the surface at 100 μL/minAntibody regeneration between different receptor dilutions was carriedout by single 5 second injections of 100 mM NaHCO₃ pH 9.4; 3M NaCl.

The same dilutions of the receptor were also injected over a BSA-FITCsurface without any ch-4-4-20 antibody at the beginning and at the endof the assay as reference injections.

Once an entire data set was collected, the resulting binding curves wereglobally fitted using computer algorithms supplied by the manufacturer,BIAcore, Inc. (Piscataway, N.J.). These algorithms calculate both the K.and K_(off), from which the apparent equilibrium binding constant, K_(D)is deduced as the ratio of the two rate constants (i.e.,K_(off)/K_(on)). More detailed treatments of how the individual rateconstants are derived can be found in the BlAevaluaion Software Handbook(BIAcore, Inc., Piscataway, N.J.).

Binding curves for two different concentrations (200 nM and 800 nM forFcγRIIIA and 200 nM and 400 nM for FcγRIIB fusion protein) were alignedand responses adjusted to the same level of captured antibodies, and thereference curves were subtracted from the experimental curves.Association and dissociation phases were fitted separately. Dissociationrate constant was obtained for interval 32-34 sec of the dissociationphase; association phase fit was obtained by a 1:1 Langmuir model andbase fit was selected on the basis R_(max) and chi² criteria.

Results

FIG. 15 shows the capture of ch 4-4-20 antibodies with mutant Fc regionson the BSA-FITC-immobilized sensor chip. 6 μL of antibodies at aconcentration of about 20 μg/mL were injected at 5 μL/min over theBSA-FITC surface. FIG. 16 is a sensogram of real time binding ofFcγRIIIA to ch-4-4-20 antibodies carrying variant Fc regions. Binding ofFcγRIIIA was analyzed at 200 nM concentration and resonance signalresponses were normalized at the level of the response obtained for thewild type ch-4-4-20 antibody. Kinetic parameters for the binding ofFcγRIIIA to ch-4-4-20 antibodies were obtained by fitting the dataobtained at two different FcγRIIIA concentrations, 200 and 800 nM (FIG.17). The solid line represents the association fit which was obtainedbased on the K_(off) values calculated for the dissociation curves ininterval 32-34 seconds. K_(D) and K_(off) represent the averagecalculated from the two different FcγRIIIA concentrations used. FIG. 18is a sensogram of real time binding of FcγRIIB-Fc fusion protein toch-4-4-20 antibodies carrying variant Fc regions. Binding of FcγRIIB-Fcfusion protein was analyzed at 200 nM concentration and resonance signalresponses were normalized at the level of the response obtained for thewild type ch-4-4-20 antibody. Kinetic parameters for the binding ofFcγRIIB-Fc fusion protein to ch-4-4-20 antibodies were obtained byfitting the data obtained at two different FcγRIIB-Fc fusion proteinconcentrations, 200 and 800 nM (FIG. 19). The solid line represents theassociation fit which was obtained based on the K_(off) calculated forthe dissociation curves in interval 32-34 seconds. K_(D) and K_(off)represent the average from the two different FcγRIIB-Fc fusion proteinconcentrations used.

The kinetic parameters (K_(on) and K_(off)) that were determined fromthe BIAcore analysis correlated with the binding characteristic of themutants as determined by an ELISA assay and the functional activity ofthe mutants as determined in an ADCC assay. Specifically, as seen inTable 19, mutants that had an enhanced ADCC activity relative to thewild-type protein, and had an enhanced binding to FcγRIIIA as determinedby an ELISA assay had an improved K_(off) for FcγRIIIA (i.e., a lowerK_(off)). Therefore, a lower K_(off) value for FcγRIIIA for a mutant Fcprotein relative to a wild type protein may be likely to have anenhanced ADCC function. On the other hand, as seen in Table 20, mutantsthat had an enhanced ADCC activity relative to the wild-type protein,and had a reduced binding for FcγRIIB-Fc fusion protein as determined byan ELISA assay had a higher K_(off) for FcγRIIB-Fc fusion protein.

Thus, the K_(off) values for FcγRIIIA and FcγRIIB can be used aspredictive measures of how a mutant will behave in a functional assaysuch as an ADCC assay. In fact, ratios of K_(off) values for FcγRIIIAand FcγRIIB-Fc fusion protein of the mutants to the wild type proteinwere plotted against ADCC data (FIG. 20). Specifically, in the case ofK_(off) values for FcγRIIIA, the ratio of K_(off) (wt) K_(off) (mutant)was plotted against the ADCC data; and in the case of K_(off) values forFcγRIIB, the ratio of K_(off) (mut)/K_(off) (wt) was plotted against theADCC data. Numbers higher than one (1) show a decreased dissociationrate for FcγRIIIA and an increased dissociation rate for FcγRIIB-Fcrelative to wild type. Mutants that fall within the indicated box have alower off rate for FcγRIIIA binding and a higher off-rate for FcγRIIB-Fcbinding, and possess an enhanced ADCC function.

TABLE 19 Kinetic parameters of FcRIIIa binding to ch4-4-20Ab obtained by“separate fit” of 200 nM and 800 nM binding curves

Highlighted mutants do not fit to the group by ELISA or ADCC data.

TABLE 20 Kinetic parameters of FcRIIB-Fc binding to wild type and mutantch4-4-20Ab obtained by “separate fit” of 200 nM and 800 nM bindingcurves.

6.9 Screening for Fc Mutants Using Multiple Rounds of Enrichment Using aSolid Phase Assay

The following mutant screens were aimed at identifying additional setsof mutants that show improved binding to FcγRIIIA and reduced binding toFcγRIIB. Secondary screening of selected Fc variants was performed byELISA followed by testing for ADCC in the 4-4-20 system. Mutants werethan selected primarily based on their ability to mediate ADCC via4-4-20 using Fluorescein coated SK-BR3 cells as targets and isolatedPBMC from human donors as the effector cell population. Fc mutants thatshowed a relative increase in ADCC, e.g., an enhancement by a factor of2 were than cloned into anti-HER2/neu or anti-CD20 chAbs and tested inan ADCC assay using the appropriate tumor cells as targets. The mutantswere also analyzed by BIAcore and their relative K_(off) weredetermined.

Screen 1: Sequential Solid Phase Depletion and Selection Using MagneticBeads Coated with FcγRIIB Followed by Selection with Magnetic BeadsCoated with FcγRIIIA.

The aim of this screen was identification of Fc mutants that either nolonger bind FcγRIIB or show reduced binding to FcγRIIB. A 10-fold excessof the naïve library (˜10⁷ cells) was incubated with magnetic beads (“MyOne”, Dynal) coated with FcγRIIB. Yeast bound to beads were separatedfrom the non-bound fraction by placing the tube containing the mixturein a magnetic field. Those yeast cells that were not bound to the beadswere removed and placed in fresh media. They were next bound to beadsthat were coated with FcγRIIIA. Yeast bound to beads were separated fromthe nonbound fraction by placing the tube containing the mixture in amagnetic field. Nonbound yeast were removed and the bound cells wereremoved by vigorous vortexing. The recovered cells were regrown inglucose containing media and reinduced in selective media containinggalactose. The selection process was repeated. The final culture wasthan used to harvest DNA. Inserts containing the Fc domain wereamplified by PCR and cloned into 4-4-20. Approximately 90 Fc mutantswere screened by 4-4-20 ELISA and ADCC assays and the resultant positivemutants are shown in Table 21.

TABLE 21 Mutants selected by sequential solid phase depletion andselection using Magnetic beads coated with FcγRIIB followed by selectionwith magnetic beads coated with FcγRIIIA. Mutant Amino Acid changesMgFc37 K248M MgFc38 K392T, P396L MgFc39 E293V, Q295E, A327T MgFc41H268N, P396LN MgFc43 Y319F, P352L, P396L MgFc42 D221E, D270E, V308A,Q311H, P396L, G402D

Screens 2&3: Mutants Selected by FACS, Equilibrium and KineticScreening:

The first library screen identified a mutation at position 396, changingthe amino acid from Proline to Leucine (P396L). This Fc variant showedincreased binding to both FcγRIIIA and FcγRIIB. A second library wasconstructed using P396L as a base line. PCR mutagenesis was used togenerate ˜10⁷ mutants each of which contained the P396L mutation andcontained additional nucleotide changes. The P396L library was screenedusing two sets of conditions.

An equilibrium screen was performed using biotinylatedFcγRIIIA-linker-avitag as a monomer, using methods already described.Approximately 10-fold excess of library (10⁸ cells) was incubated in a0.5 mL of approximately 7 nM FcγRIIIA for 1 hr. The mixture was sortedby FACS, selecting top 1.2% of binders. Selected yeast cells were grownin selective media containing glucose and reinduced in selective mediacontaining galactose. The equilibrium screen was repeated a second timeand the sort gate was set to collect the top 0.2% of binders. Theselected yeast cells were then grown under selective conditions inglucose. This culture was than used to harvest DNA. Inserts containingthe Fc domain were amplified by PCR and cloned into the nucleotidesequence encoding 4-4-20 variable domain using methods alreadydescribed. Approximately 90 Fc mutants were screened by 4-4-20 ELISA andADCC and the resultant positive mutants are shown in Table 22.

TABLE 22 Mutants selected by FACS using an Equilibrium screen withconcentrations of FcRIIIA of approximately 7 nM. Mutant Amino Acidchanges MgFc43b K288R, T307A, K344E, P396L MgFc44 K334N, P396L MgFc46P217S, P396L MgFc47 K210M, P396L MgFc48 V379M, P396L MgFc49 K261N,K210M, P396L MgFc60 P217S, P396L

A kinetic screen was also implemented to identify mutants with improvedK_(off) in binding FcγRIIIA Conditions were established for screeningthe P396L library using a strain with the P396L Fc variant displayed onthe yeast surface. Briefly cells grown under inducing conditions wereincubated with 0.1 μM biotinylated FcγRIIIA-linker-avitag monomer for 1hr. The cells were washed to remove the labeled ligand. Labeled cellswere then incubated for different times with 0.1 μM unlabeledFcγRIIIA-linker-avitag monomer, washed and then stained with SA:PE forFACS analysis (FIG. 21). Cells were also stained with goat anti-human Fcto show that the Fc display was maintained during the experiment.

Based on the competition study it was determined that a 1 minuteincubation resulted in approximately 50% loss of cell staining. Thistime point was chosen for the kinetic screen using the P396L library.Approximately 10-fold excess of library (10⁸ cells) was incubated with0.1 nM biotinylated FcγRIIIA-linker-avitag monomer in a 0.5 mL volume.Cells were washed and then incubated for 1 minute with unlabeled ligand.Subsequently the cells were washed and labeled with SA:PE. The mixturewas sorted by FACS, selecting the top 0.3% of binders. Selected yeastcells were grown in selective media containing glucose and reinduced inselective media containing galactose. The kinetic screen was repeated asecond time and the sort gate was set to collect the top 0.2% ofbinders. The nonselcted P396L library was compared to the yeast cellsselected for improved binding by FACS (FIG. 22). The histograms show thepercentage of cells that are costained with both FcγRIIIA/PE and goatanti-human Fc/FITC (upper right).

The selected yeast cells from the second sort were then grown underselective conditions in glucose. This culture was than used to harvestDNA. Inserts containing the Fc domain were amplified by PCR and clonedinto the nucleotide sequence encoding 4-4-20 variable domain usingmethods described above. Approximately 90 Fc mutants were screened by4-4-20 ELISA and ADCC and the resultant positive mutants are shown inTable 23.

TABLE 23 Mutants selected by FACS using a Kinetic screen using equimolaramounts of unlabeled CD16A for 1 minute. Mutants Amino Acid changesMgFc50 P247S, P396L MgFc51 Q419H, P396L MgFc52 V240A, P396L MgFc53L410H, P396L MgFc54 F243L, V305I, A378D, F404S, P396L MgFc55 R255l,P396L MgFc57 L242F, P396L MgFc59 K370E, P396L

Screens 4 and 5: Combining the Solid Phase FcγRIIB Depletion Step withFcγRIIIA Selection by FACs Sort, Using the FcγRIIIA 158V Allele

Analysis of Fc variants from Screen 1 showed that the mutations thatwere selected from the secondary screen had improved binding to bothFcγRIIIA and FcγRIIB. Therefore, the data suggested that sequentialdepletion and selection using magnetic beads (solid phase) under theestablished conditions did not efficiently select for differentialbinding of FcγRIIIA and FcγRIIB. Therefore, in order to screen moreeffectively for mutants that bind FcγRIIIA, while having reduced or nobinding to FcγRIIB, the solid phase FcγRIIB depletion step was combinedwith FcγRIIIA selection by FACs sort. This combination identified Fcvariants that bind FcγRIIIA with greater or equal affinity thanwild-type Fc.

A 10-fold excess of the naïve library (˜10⁷) was incubated with magneticbeads coated with FcγRIIB. Yeast bound to beads were separated from thenon-bound fraction by placing the tube containing the mixture in amagnetic field. Those yeast cells that were not bound to the beads wereremoved and placed in fresh media and subsequently reinduced in mediacontaining galactose. The FcγRIIB depletion by magnetic beads wasrepeated 5 times. The resulting yeast population was analyzed and foundto show greater than 50% cell staining with goat anti-human Fc and avery small percentage of cells were stained with FcγRIIIA. These cellswere then selected twice by a FACS sort using 0.1 μM biotinylatedFcγRIIIA linker-avitag (data not shown). The FcγRIIIA was the 158Vallotype. Yeast cells were analyzed for both FcγRIIIA and FcγRIIBbinding after each sort and compared to binding by wild-type Fc domain(FIGS. 23 A-L).

The selected yeast cells from the second sort were then grown underselective conditions in glucose. This culture was then used to harvestDNA. Inserts containing the Fc domain were amplified by PCR and clonedinto the nucleotide sequence encoding 4-4-20 variable domain.Approximately 90 Fc mutants were screened by 4-4-20 ELISA and ADCC andthe resultant positive mutants are shown in Table 24 (mutants 61-66).

TABLE 24 Mutants selected by magnetic bead depletion using beads coatedwith CD32B and final selection by FACS using FcγRIIIA 158Valine or158Phenylalanine Mutants Amino Acid Changes MgFc61 A330V MgFc62 R292GMgFc63 S298N, K360R, N361D MgFc64 E233G MgFc65 N276Y MgFc66 A330V, V427MMgFc67 V284M, S298N, K334E, R355W, R416T

Screening of Fc Mutants Using the 158F allele of FcγRIIIA:

Two different alleles of FcγRIIIA receptor exist that have differentbinding affinities for the IgG1 Fc domain (Koene et al., 1997, Blood 90:1109-1114; Wu et al., 1997, J. Clin. Invest. 100: 1059-70). The 158Fallele binds to the Fc domain with a binding constant 5-10 fold lowerthan the 158V allele. Previously all of the Fc screens using yeastdisplay were done using the high binding 158V allele as a ligand. Inthis experiment, Fc mutants were selected from the FcγRIIB depletedyeast population using biotinylated FcγRIIIA158F-linker-avitag monomeras a ligand. The sort gate was set to select the top 0.25 percentFcγRIIIA 158F binders. The resulting enriched population was analyzed byFACS (FIG. 23B). Individual clones were then isolated and their bindingto different FcγRs were analyzed by FACS (FIG. 23B). Analysis ofindividual clones from the population resulted in the identification ofa single mutant harboring 5 mutations MgFc67 (V284M, S298N, K334E,R355W, R416S), which had an enhanced binding to FcγRIIIA and a reducedbinding to FcγRIIB.

Secondary Screen of Mutants by an ADCC Assay for Screens 1, 2, and 3:

Mutants that were selected in the above screens were then analyzed usinga standard ADCC assay to determine the relative rates of lysis mediatedby ch4-4-20 harboring the Fc mutants. ch4-4-20 antibodies carrying theFc variants were constructed using methods already described above.SK-BR3 cells were used as targets and effector cells were PBMC that wereisolated from donors using a Ficoll gradient, as described supra(Section 6.7). The ADCC activity results for the mutants are summarizedin Table 25.

As seen in Table 25, mutants isolated using the above primary andsecondary screens based on FcγRIIB depletion and FcγRIIIA selectionshowed enhanced ADCC activity relative to wild-type.

TABLE 25 Analysis of ADCC mediated by 4-4-20 anti-Fluorescein antibodyon SKBR3 cells coated with fluorescein. Relative rate of Mutant AminoAcid Change lysis MgFc37 K248M 3.83 MgFc38 K392T, P396L 3.07 MgFc39E293V, Q295E, A327T 4.29 MgFc41 H268N, P396LN 2.24 MgFc43 Y319F, P352L,P396L 1.09 MgFc42 D221E, D270E, V308A, Q311H, P396L, 3.17 G402D MgFc43bK288R, T307A, K344E, P396L 3.3 MgFc44 K334N, P396L 2.43 MgFc46 P217S,P396L 2.04 MgFc47 K210M, P396L 2.02 MgFc48 V379M, P396L 2.01 MgFc49K261N, K210M, P396L 2.06 MgFc50 P247S, P396L 2.1 MgFc51 Q419H, P396L2.24 MgFc52 V240A, P396L 2.35 MgFc53 L410H, P396L 2 MgFc54 F243L, V305I,A378D, F404S, P396L 3.59 MgFc55 R255l, P396L 2.79 MgFc57 L242F, P396L2.4 MgFc59 K370E, P396L 2.47 MgFc60 P217S, P396L 1.44

Mutants 37, 38, 39, 41, 43 were analyzed using 0.5 μg/mL ch4-4-20. Allother antibodies were tested at 1 μg/mL. All rates were normalized towild type ch4-4-20 (IgG1).

Mutants were additionally cloned into the heavy chain of antitumormonoclonal antibody 4D5 (anti-HER2/neu) and anti-CD20 monoclonalantibody 2H7 by replacing the Fc domain of these monoclonal antibodies.These chimeric monoclonal antibodies were expressed and purified andtested in an ADCC assay using standard methods by transient transfectioninto 293H cells and purification over protein G column. The chimeric 4D5antibodies were tested in an ADCC assay using SK-BR3 cells as targets(FIG. 24), whereas the chimeric 2H7 antibodies were tested in an ADCCassay using Daudi cells as targets (FIG. 25).

Secondary Screen of Mutants Via BIAcore:

Mutants that were selected in the above screens were then analyzed byBIAcore to determine the kinetic parameters for binding FcγRIIIA(158V)and FcγRIIB. The method used was similar to that disclosed in Section6.8, supra.

The data displayed are K_(off) values relative to wild type off rates asdetermined from experiments using the Fc mutants in the ch4-4-20monoclonal antibody. Relative numbers greater than one indicate adecrease in K_(off) rate. Numbers less than one indicate an increase inoff rate.

Mutants that showed a decrease in off rates for FcγRIIIA were MgFc38(K392, P396L), MgFc43(Y319F, P352L, P396L), MgFc42(D221E, D270E, V308A,Q311H, P396L, G402D), MgFc43b (K288R, T307A, K344E, P396L), MgFc44(K334N, P396L), MgFc46 (P217S, P396L), MgFc49 (K261N, K210M, P396L).Mutants that showed a decrease in off rate for FcγRIIB were,MgFc38(K392, P396L), MgFc39 (E293V, Q295E, A327T), MgFc43 (K288R, T307A,K344E, P396L), MgFc44 (K334N, P396L). The Biacore data is summarized inTable 26.

TABLE 26 BIAcore data. FcγRIIIA FcγRIIB Fc 158V (Koff WT/ mutant AAresidues (Koff WT/Mut) Mut) MgFc37 K248M 0.977 1.03 MgFc38 K392T, P396L1.64 2.3 MgFc39 E293V, Q295E, A327T 0.86 1.3 MgFc41 H268N, P396LN 0.921.04 MgFc43 Y319F, P352L, P396L 1.23 2.29 MgFc42 D221E, D270E, V308A,1.38 Q311H, P396L, G402D MgFc43b K288R, T307A, K344E, P396L 1.27 0.89MgFc44 K334N, P396L 1.27 1.33 MgFc46 P217S, P396L 1.17 0.95 MgFc47K210M, P396L MgFc48 V379M, P396L MgFc49 K261N, K210M, P396L 1.29 0.85MgFc50 P247S, P396L MgFc51 Q419H, P396L MgFc52 V240A, P396L MgFc53L410H, P396L MgFc54 F243L, V305I, A378D, F404S, P396L MgFc55 R255l,P396L MgFc57 L242F, P396L MgFc59 K370E, P396L MgFc60 P217S, P396L MgFc61A330V 1 0.61 MgFc62 R292G 1 0.67 MgFc63 S298N, K360R, N361D 1 0.67MgFc64 E233G 1 0.54 MgFc65 N276Y 1 0.64 MgFc66 A330V, G427M, 1 0.62MgFc67 V284M, S298N, K334E, R355W, R416T

6.10 PBMC Mediated ADCC Assays

Materials and Methods

Fc variants that show improved binding to FcγRIIIA were tested by PBMCbased ADCC using 60:1 effector:target ratio. Two different tumor modelsystems were used as targets, SK-BR3 (anti-HER2/neu) and Daudi(anti-CD20). Percent specific Lysis was quantitated for each mutant.Linear regression analysis was used to plot the data setting the maximalpercent lysis at 100%.

ADCC is activated on immune system effector cells via a signaltransduction pathway that is triggered by an interaction between lowaffinity FcγR and an immune complex. Effector cell populations werederived from either primary blood or activated monocyte derivedmacrophages (MDM). Target cells were loaded with europium and incubatedwith chimeric MAb and subsequently incubated with effector cellpopulations. Europium works the same way as ⁵¹Cr, but it isnon-radioactive and the released europium is detected in a fluorescentplate reader. Lymphocytes harvested from peripheral blood of donors(PBM) using a Ficoll-Paque gradient (Pharmacia) contain primarilynatural killer cells (NK). The majority of the ADCC activity will occurvia the NK containing FcγRIIIA but not FcγRIIB on their surface.

Experiments were performed using two different target cell populations,SK-BR-3 and Daudi, expressing HER2/neu and CD20, respectively. ADCCassays were set up using Ch4-4-20/FITC coated SK-BR-3, Ch4D5/SKBR3, andRituxan/Daudi (data not shown). Chimeric MAbs were modified using Fcmutations identified. Fc mutants were cloned into Ch4D5. Purified Ab wasused to opsonize SK-BR-3 cells or Daudi cells. Fc mutants were clonedinto Ch4D5.

Results.

Fc mutants showed improved PBMC mediated ADCC activity in SK BR3 cells(FIG. 28). The plot shows linear regression analysis of a standard ADCCassay. Antibody was titrated over 3 logs using an effector to targetratio of 75:1. % lysis=(Experimental release—SR)/(MR−SR)*100.

Fc mutants showed improved PBMC mediated ADCC activity in Daudi cells(FIG. 29).

6.11 Monocyte Derived Macrophage (MDM) Based ADCC Assays

FcγR dependent tumor cell killing is mediated by macrophage and NK cellsin mouse tumor models (Clynes et al., 1998, PNAS USA, 95: 652-6).Elutriated monocytes from donors were used as effector cells to analyzethe efficiency Fc mutants to trigger cell cytotoxicity of target cellsin ADCC assays. Expression patterns of FcγRI, FcγR3A, and FcγR2B areaffected by different growth conditions. FcγR expression from frozenmonocytes cultured in media containing different combinations ofcytokines and human serum were examined by FACS using FcR specific MAbs.(FIGS. 30A-30O). Cultured cells were stained with FcγR specificantibodies and analyzed by FACS to determine MDM FcγR profiles.Conditions that best mimic macrophage in vivo FcγR expression, i.e.,showed the greatest fraction of cells expressing CD16 and CD32B wereused in a monocyte derived macrophage (MDM) based ADCC assay. For theexperiment in FIGS. 30A-30O, frozen elutriated monocytes were grown for8 days in DMEM and 20% FBS containing either M-CSF (condition 1) orGM-CSF (condition 2). For the experiment in FIG. 31, frozen elutriatedmonocytes were cultured for 2 days in DMEM and 20% FBS containingGM-CSF, IL-2 and IFNγ prior to ADCC assay. Serum free conditions havealso been developed which allow for high levels of CD16 and CD32Bexpression (data not shown). Briefly, purified monocytes were grown for6-8 days in Macrophage-SFM (Invitrogen) containing GM-CSF, M-CSF, IL-6,IL-10, and IL-1β. While the incidence of CD32B+/CD16+ cells in thesecultures is highest using a mixture of cytokines, combinations of two ofmore cytokines will also enhance FcγR expression (M-CSF/IL-6,M-CSF/IL-10; or M-CSF/IL-1β). For ADCC assays, IFNγ is added for thefinal 24-48 hours.

MDM based ADCC required incubation times of >16 hrs to observe targetcell killing. Target cells were loaded with Indium-111 which is retainedfor long incubations within the target cells. Indium release wasquantitated using a gamma counter. All other reagents, Abs and targetcells, were similar to the PBMC based ADCC assay. ADCC activity due toFcγRI can be efficiently blocked using the anti-FcRI blocking antibody(M21, Ancell). The assay conditions differ slightly from the PBMC basedassay. 20:1 target to effector; 18-14 hr incubation at 37C.

Fc mutants that show improved PBMC ADCC, increased binding to FcγRIIIA,or decreased binding to FcγRIIB were tested (FIG. 31).

6.12 Effect of Fc Mutants on Complement Activity

Fc mutants were originally identified based on their increased bindingto FcγRIIIA. These mutants were subsequently validated for theirimproved affinity for all low affinity receptors and in many casesimproved activity in ADCC mediated by PBMC. In vivo antibody mediatedcytotoxicity can occur through multiple mechanisms. In addition to ADCCother possible mechanisms include complement dependent cytotoxicity(CDC) and apoptosis. The binding of C1q to the Fc region of animmunoglobulin initiates as cascade resulting in cell lysis by CDC. Theinteraction between C1q and the Fc has been studies in a series of Fcmutants. The results of these experiments indicate that C1q and the lowaffinity FcR bind to overlapping regions of the Fc, however the exactcontact residues within the Fc vary.

Mutants that showed improved ADCC in the PBMC based assay were examinedfor their effect in CDC. Antibodies were analyzed in the anti CD20Ch-mAb, 2H7. We detected improved CDC for each mutant ch-mAb tested.Interestingly even though these mutants were selected for their improvedADCC they also show enhanced CDC

Materials and Methods.

CDC assay was used to test the Fc mutants using anti-CD20 and Daudicells as targets. Guinea Pig Serum was used as the source for complement(US Biological). The CDC assay was similar to PBMC based ADCC. Targetcells were loaded with europium and opsonized with ChMAb. Howevercomplement, guinea pig serum, was added instead of effector cells. FIG.32 shows a flow chart of the assay. Anti-CD20 ChMab over 3 orders ofmagnitude was titrated. % lysis was calculated. Daudi cells, (3×10⁶)were labeled with BADTA reagent. 1×10⁴ cells were aliquoted into wellsin a 96 well plate. Antibodies were titrated into the wells using 3 folddilutions. The opsonization reaction was allowed to proceed for 30-40minutes at 37° C. in 5% CO₂. Guinea pig serum was added to a final conc.of 20%. The reaction was allowed to proceed for 3.5 hrs at 37° C. in 5%CO₂. Subsequently, 100 uls of cell media was added to the reaction andcells were spun down. For detection 20 uls of the supernatant was addedto 200 uls of the Europium solution and the plates were read in theVictor2(Wallac).

Results:

All mutants that show improved binding for either activating FcR or C1qwere placed in the CDC assay (FIG. 33). Fc mutants that showed enhancedbinding to FcγRIIIA also showed improved complement activity Each of themutants show enhanced complement activity compared to wild type. Themutants tested are double mutants. In each case one of the mutationspresent is P396L.

To determine whether the increase in CDC correlated with increasedbinding of C1q to IgG1 Fc binding between the two proteins was measuredin realtime using surface plasmon resonance. In order to examine thebinding between C1q and an IgG1 Fc the Fc variants were cloned into ananti-CD32B ch-mAb, 2B6. This allowed us to capture the wt and mutantantibodies to the glass slide via soluble CD32B protein (FIG. 35A).Three of the four mutants tested in CDC were also examined in theBiacore. All 3 showed greatly enhanced K_(off) compare to wild type Fc(FIG. 35B). Biacore format for C1q binding to 2B6 mutants demonstrateenhanced binding of mutants with P396L mutation (FIG. 36). MutationD270E can reduce C1q binding at different extent. A summary of thekinetic analysis of FcγR and C1q binding is depicted in the Table 27below.

TABLE 27 KINETIC ANALYSIS OF FcgR and C1q binding to mutant 2B62B6Mutants 3aV158 3aF158 2bfcagl 2aR131Fcagl 2aH131Fcagl C1q WT 0.1920.434 0.056 0.070 0.053 0.124 MgFc38 0.114 0.243 0.024 0.028 0.024 0.096(K392T, P396L) MgFc51 0.142 0.310 0.030 0.036 0.028 0.074 (Q419H, P396L)MgFc55 0.146 0.330 0.030 0.034 0.028 0.080 (R255I, P396L) MgFc59 0.1490.338 0.028 0.033 0.028 0.078 (K370E, P396L) MgFc31/60 0.084 0.238 0.0940.127 0.034 0.210 MgFc51/60 0.112 0.293 0.077 0.089 0.028 0.079MgFc55/60 0.113 0.288 0.078 0.099 0.025 0.108 MgFc59/60 0.105 0.2960.078 0.095 0.024 0.107

6.13 Designing Fc Variants with Decreased Binding to FcγRIIB

Based on a selection for Fc mutants that reduce binding to FcγRIIB andincrease binding to FcγRIIA 131H a number of mutations including D270Ewas identified. Each mutation was tested individually for binding to thelow affinity Fc receptors and their allelic variants by Biacoreperformed as described above in the context of the 4D5 ChAb(anti-HER2/neu).

D270E had the binding characteristics that suggested it wouldspecifically reduce FcγRIIB binding. D270E was tested in combinationwith mutations that were previously identified based on their improvedbinding to all FcR.

Results.

As shown in Tables 28 and 29 and FIGS. 37 and 38 addition of D270Emutation enhances FcγRIIIA and FcγRIIA H131 binding and reduces bindingto FcγRIIB. FIG. 39 shows the plot of MDM ADCC data against the Koff asdetermined for CD32A H131H binding for select mutants.

TABLE 28 ADDITION OF D270E MUTATION ENHANCES FcγRIIIA AND FcγRIIA H131BINDING AND REDUCES FcγRIIB BINDING 4D5Mutants 3aV158 3aF158 2bfcagl2aR131Fcagl 2aH131Fcagl Wt pure 0.175 0.408 0.078 0.067 0.046 MgFc550.148 0.381 0.036 0.033 0.029 MgFc55/60 0.120 0.320 0.092 0.087 0.013MgFc55/60 + 0.116 0.405 0.124 0.112 0.037 R292G MgFc55/60 + 0.106 0.3040.092 0.087 0.015 Y300L MgFc52 0.140 0.359 0.038 0.040 0.026 MgFc52/600.122 0.315 0.094 0.087 0.013 MgFc59 0.145 0.378 0.039 0.047 0.033MgFc59/60 0.117 0.273 0.088 0.082 0.012 MgFc31 0.125 0.305 0.040 0.0430.030 MgFc31/60 0.085 0.215 0.139 0.132 0.020 MgFc51 0.135 0.442 0.0600.047 0.062 MgFc51/60 0.098 0.264 0.118 0.106 0.023 MgFc38 0.108 0.2920.034 0.025 0.032 MgFc38/60 0.089 0.232 0.101 0.093 0.021 D265A n.b.n.b. n.b. 0.223 0.117

TABLE 29 KINETIC CHARACTERISTICS OF 4D5 MUTANTS 4D5Mutants 3aV158 3aF1582bfcagl 2aR131Fcagl 2aH131Fcagl MgFc70 0.101 0.250 0.030 0.025 0.025MgFc71 0.074 0.212 0.102 0.094 0.020 MgFc73 0.132 0.306 0.190 — 0.024MgFc74 0.063 0.370 n.b. 0.311 0.166 WT023stable 0.150 0.419 0.071 0.0680.043

6.14 Ability to Mediate Cell Lysis by ADCC and CDC Correlates withEnhancement of Functional Range of the Antibody

Fc mutations which enhance FcγRIIIA and FcγRIIA binding and reducebinding to FcγRIIB have been suggested to positively correlate with theappearance or improvement of both ADCC and complement function (Section6.8). This hypothesis was tested by cloning promising mutations into theheavy chain of the chimeric antitumor monoclonal antibody 4D5(anti-HER2/neu), chimeric anti-CD32B monoclonal antibody ch2B6 and theanti-CD20 antibody RITUXIN™ anti-CD-20 antibody. Mutations were clonedinto the heavy chains of the antibodies using standard techniques. Thesechimeric antibodies were expressed by transient transfection into 293Hcells and purified over a protein G column. Variant 4D5 antibodies wereanalyzed for alterations in kinetic parameters using a BIAcore assay(BIAcore instrument 1000, BIAcore Inc., Piscataway, N.J.) and associatedsoftware as described supra (Section 6.8). Binding ability of 4D5 andch2B6 antibodies was characterized by immunostaining cells with eitherFITC conjugated variant antibody or the variant antibodies and aPE-conjugated polyclonal F(ab)₂ goat anti-human Fc antibody (JacksonImmunoresearch Laboratories, Inc.). FACS analysis was used to quantitatethe staining.

The chimeric variant antibodies were tested in an ADCC or CDC assay asdescribed supra (Section 6.10 and 6.11, respectively). For both 4D5 andch2B6 antibodies, the effects of antigen density on binding or on celllysis by ADCC/CDC were tested by using cells with high or low expressionof antigen. Antigen density was determined using QUANTUM™ SIMPLYCELLULAR® kit from Bangs Laboratories, Inc. (Fishers, Ind.) according tothe manufacturer's instructions. Target cells for 4D5 antibodies wereSK-BR3 cells (high Her2/neu expression) and HT29 cells (low Her2/neuexpression). Target cells for ch2B6 antibodies were Daudi cells or BL41cells (high CD32B expression) and Ramos cells (low CD32B expression).Target cells for RITUXIN™ anti-CD-20 antibody were CHO cells, which wereengineered to express both CD32B and CD20 using standard techniques.

Results

FIGS. 40 and 41 show the capture of 4D5 antibodies with mutant Fcregions on the BSA-FITC-immobilized sensor chip. BIAcore data wasanalyzed as described in Section 6.8. Either triple mutants (FIG. 40) orquadruple mutants (FIG. 41) showed reduced K_(d) to the activating Fcreceptors and increased K_(d) to the inhibitory Fc receptor.

Although the Fc mutant 31/60 (P247L; N421K; D270E) did not enhance 4D5binding to cells expressing low levels of Her2/neu (FIG. 42), thismodification, as well as variants 71 (D270E; G316D; R416G), 59/60(K370E; P396L; D270E), 55/60 (R255L; P396L; D270E), 51/60 (Q419H; P396L;D270E), 55/60/F243L (R255L; P396L; D270E; F243L), and 74/P396L (F243L;R292P; V3051; P396L) improved the wild-type ADCC mediated lysis of cellsexpressing low levels of antigen (FIGS. 43 and 44).

When similar Fc mutations, variants 31/60, 59/60, and 71, are introducedinto an antibody with only limited binding to cells expressing lowlevels of antigen and no native effector function on the same cells, theresults are more dramatic. FIGS. 45A-45H demonstrate that wild-typech2B6 binding to Ramos cells can be substantially improved by theintroduction the Fc mutations of variant 31/60 and 59/60. Similarly,effector function can be introduced by Fc mutations. Where the wild-typeantibody has no detectable effector function, Fc mutations can result ina gain-of-function phenotype. Mutations which improved the binding ofch2B6 to Ramos cells also enabled the mutated antibody to mediate ADCC,variant 31/60, or CDC, variants 31/60 and 71 (FIGS. 46 and 47,respectively). FIGS. 48 and 49 also show the spectrum of responseavailable, dependent on the specific mutation. Where the wild type ch2B6antibody is capable of mediating at least some effector function, e.g.in cells with high expression of CD32B, Daudi cells, the same Fcmutations, variant 31/60 and 71, improve the effect (FIG. 48).

The increase in ADCC activity was shown to be a function of the Fcmodification and not the target antigen. The mutation variant 55/60,previously identified as improving ADCC activity in 4D5 antibody,conferred effector function to the anti-CD20 antibody, RITUXIN™anti-CD20 antibody. FIGS. 49 A and B show that the engineered CHO cellline expressed similar levels of CD32B and CD20 when tested withFITC-conjugated 2B6 or RITUXIN™ anti-CD20 antibody, respectively.Although the cells were sensitive to ADCC mediated by wild-type 2B6,ADCC activity was completely undetectable using wild-type RITUXIN™anti-CD20 antibody (FIG. 50 A). The introduction of the mutation variant55/60 into RITUXIN™ anti-CD20 antibody, as in 4D5, was, however, able toconfer effector function to the modified antibody (FIG. 50 B).

Possible mechanisms by which the mutated antibodies were able to improveboth binding and effector function were observed when the bindingaffinities of variant ch2B6 antibodies to FcγRIIB were correlated withtheir ability to bind Ramos cells (FIG. 51A-B). For example, variant55/60 had both the highest k_(off) and binding affinity to Ramos cells.It is theorized the limited ability of the wild-type antibody to bindFcγRIIB is due to Fc-FcγRIIB interaction, effectively withdrawing theadditional cell surface receptors from further antibody binding. Thetheory was investigated by challenging opsonized Ramos cells with CD16A,an activating FcγR. In accord with the theory, at low antigen density,Fc-engineered ch2B6, but not wild type Fc, was able bind the activatingreceptor (FIG. 52).

6.15 Effect of Light-Chain Glcosylation on Fc Binding to FcγR andMediation of ADCC and Compliment Activity

As demonstrated in Sections 6.8 and 6.14, Fc mutations can introduce orimprove both the binding and effector function of antibodies. The effectof light-chain glycosylation on these abilities was investigated bycombining a mutation which eliminated glycosylation of the light-chainregion (YA substitution at positions 50 and 51 of the light-chain aminoacid sequence) with the previously identified Fc mutations which inducedor improved ADCC or complement function as listed in Table 30.

Mutations were cloned into both the heavy and light chains of anti-FcγRmonoclonal antibody ch2B6. These chimeric antibodies were expresses andpurified using standard methods by transient transfection into 293Hcells and purification over a protein G column. Variant antibodies wereanalyzed for alterations in kinetic parameters using a BIAcore assay(BIAcore instrument 1000, BIAcore Inc., Piscataway, N.J.) and associatedsoftware as described supra (Section 6.8). Binding ability wascharacterized by immunostaining cells with the variant antibodies and aPE-conjugated polyclonal F(ab)₂ goat anti-human FcγR antibody (JacksonImmunoresearch Laboratories, Inc.). FACS analysis was used to quantitatethe staining The effects of antigen density were investigated byimmunostaining high (Daudi cells and EL-4/CD32B cells) and low (Ramos)antigen expressing cells.

The chimeric variant antibodies were tested in an ADCC or CDC assayusing Ramos cells as targets as described supra (Section 6.10 and 6.11,respectively).

Results

BIAcore analysis revealed that previously identified mutations whichenhanced FcγRIIIA binding and reduced binding to FcγRIIB were unaffectedby the glycosylation state of the light chain (Table 30).

TABLE 30 ADDITION OF MUTATION ELIMINATING LIGHT-CHAIN GLYCOSYLATION ATPOSITION 50 DOES NOT AFFECT IMPROVED BINDING TO FcγRIIIA AND DECREASEDBINDING TO FcγRIIB 3A 3A 2B 2A 2A Antibody VL Fc Date V158 F158 FcaglyR131 H131 Ch2B6 Mouse-Wt- WT 0.150 0.427 0.074 0.071 0.043 gly. position50 Ch2B6 Mouse-Wt- agly nb nb nb nb nb agly gly. position 50 Ch2B6Mouse-Wt- Q419H; Mar. 15, 2004 0.098 0.264 0.118 0.106 0.023 51/60 gly.position P396L; 50 D270E Ch2B6 Mouse-Wt- P247L; Mar. 15, 2004 0.0840.245 0.156 0.141 0.027 31/60 gly. position N421K; 50 D270E Ch2b6Mouse-Wt- R255L; Mar. 15, 2004 0.120 0.320 0.092 0.087 0.013 55/60 gly.position P396L; 50 D270E hu2B6YA Human -agly Q419H; Mar. 15, 2004 0.0980.264 0.118 0.106 0.023 51/60 YA P396L; substitution D270E at pos. 50,51 hu2B6YA Human - agly P247L; Mar. 15, 2004 0.084 0.245 0.156 0.1410.027 31/60 YA N421K; substitution D270E at pos. 50, 51 hu2B6YA Human -agly R255L; Mar. 15, 2004 0.120 0.320 0.092 0.087 0.013 55/60 YA P396L;substitution D270E at pos. 50, 51s hu2B6 human-Wt- WT 0.150 0.427 0.0740.071 0.043 WT gly. position 50

The improvements in mutant antibody binding noted in the BIAcoreanalysis were not observed when cells with high antigen expression wereimmunostained and analyzed by FACS (FIGS. 53 and 54). Variant antibodiesfailed to improve binding, exhibiting characteristics similar to thewild type antibody. However, when the Ramos cell line was tested,wild-type ch2B6 and ch2B6 variant 31/60 bound at levels significantlyabove that of the other variants tested (FIG. 55). As the immunostainingprotocol more closely approximates the in vivo conditions ofantigen-antibody interaction, the differences in the two analyses maysuggest that light-chain glycosylation is necessary for biologicallyrelevant activity of the antibody.

This suggestion was furthered by the ADCC and CDC assays. Although theseFc mutations resulted in a gain-of-function for 4D5 antibodies asoutlined in Section 6.14, for ch2B6, the ability to effect lysis wasdependent on the glycosylation state of the light-chain of the antibody(FIGS. 56 and 57).

6.16 Effect of Mutations Identified as Enhancing ADCC Function in ADCCAssays Using Tumor Cells Isolated from Rituxan™ Anti-CD-20 AntibodyTreated Patients

Fc mutations which enhance FcγRIIIA and FcγRIIA binding, reduce bindingto FcγRIIB and enhance ADCC and/or complement function (Section 6.8)were cloned into the anti-CD20 antibody RITUXIN™ anti-CD20 antibodyusing standard techniques. These chimeric antibodies were expressed bytransient transfection into 293H cells and purified over a protein Gcolumn. The variant antibodies were tested in an ADCC or CDC assay asdescribed supra (Section 6.10 and 6.11, respectively) in cells isolatedfrom RITUXIN™ anti-CD20 antibody treated patients.

During the course of phase I and phase II clinical trials of Rituximab,lymphoma cells from biopsy specimens obtained from patients with B celllymphoma prior to receiving the antibody were collected. Participatingpatients underwent surgical removal of a lymph node near the surface ofthe body. This was done using a local anesthetic. A portion of thetissue was analyzed by routine histopathology in the pathology lab. Aportion of the lymph node was used to make a cell suspension for the invitro studies.

Additionally, pre- and post-treatment PBMC via leukapheresis in some ofthe patients were collected to study the effector cells and T cellimmune response after Rituximab treatment. Peripheral blood T cells andeffector cells were collected via leukapheresis from patients treatedwith Rituximab. Participating patients underwent leukapheresis beforethe Rituximab treatment and one month after completion of the treatmentto collect the T lymphocytes and effector cells. The collected bloodcomponents were mixed with an anti-coagulant (ACD-A) as it was drawn toprevent clotting. The effector cells collected via leukapheresis wereused to determine if effector cells of different FcγR genotypes mediateADCC differently.

Results

The results of the ADCC assays for the different Fc Engineered rituximabantibodies in six of the patients are shown in FIGS. 58A-F. Tables 31and 32 provide a ranking of the effectiveness of the antibodies in sixpatients with 1 being the most effective for that patient and 11 beingthe least effective for that patient. A normal donor provided PBMC forthis experiment. The genotype of the normal donor was heterozygous forthe FcRIIIA 158V and FcRIIA 131R alleles. In most patients, the Fcengineered rituximab antibodies showed an improvement over rituximab inADCC activity.

TABLE 31 (10:1 Effector:Target Ratio) Fc Mutant IgG1 Rituximab55/60/300L 51/60 52/60 59/60 38/60 59 51 31/60 55/60/292G Patient 1 1110 5 3 4 1 9 8 7 6 2 Patient 2 11 9 2 10 4 1 7 3 6 5 8 Patient 3 11 10 34 8 2 9 5 7 6 1 Patient 4 11 9 1 6 8 5 10 7 3 4 2 Patient 5 11 7 8 10 21 9 3 6 5 4 Patient 6 11 10 8 4 1 2 6 5 9 7 2

TABLE 32 (30:1 Effector:Target Ratio) Fc Mutant IgGl Rituximab55/60/300L 51/60 52/60 59/60 38/60 59 51 31/60 55/60/292G Patient 1 1110 6 7 8 2 9 4 5 3 1 Patient 2 11 8 1 4 5 2 6 3 10 7 9 Patient 3 11 8 21 3 6 7 5 10 4 10 Patient 4 11 5 1 2 9 3 8 6 10 4 7 Patient 5 11 9 2 5 61 10 4 8 3 7 Patient 6 11 10 6 8 4 1 2 3 9 5 7

As shown in FIG. 58 A, rituximab has minimal ADCC killing activity ascompared to the other engineered rituximab antibodies tested. Patient 1fits our definition of a non-responder (i.e., is refractory) torituximab treatment (FIG. 58 A). In contrast, in patient 2, wild-typerituximab shows some ADCC activity; however all tested variants except59/60 and 52/60 exhibited improved ADCC activity.

6.17 Efficacy in a Mouse Model

Efficacy of Fc engineered rituximab antibodies may be investigated in amouse xenographic model (nu/nu mice, female, approximately 10 weeks old)utilizing a B cell lymphoblastic tumor, e.g., Ramos cell tumors. Ramoscells (ATCC, CRL 1596) are maintained in culture using RPMI-1640supplemented with 10% fetal calf serum and glutamine at 37° C. and 5%CO₂. To increase tumorigenicity of the cell line, Tumors are firstinitiated in female nude mice approximately 7-10 weeks old bysubcutaneous injection of 1.7×10⁶ Ramos cells in a volume of 0.10 ml(HBSS) using a 1 cc syringe fitted with 25 g needle. All animals aremanipulated in a laminar flow hood and all cages, bedding, food andwater are autoclaved. Tumor cells are passaged by excising tumors andpassing these through a 40 mesh screen; cells are washed twice with1×HBSS (50 ml) by centrifugation (1300 RPM), resuspended in 1×HBSS to10×10⁶ cells/ml, and frozen at −70° C. until used.

The one-passaged tumor cells from several frozen lots are thawed,pelleted by centrifugation (1300 RPM), washed twice with 1×HBSS, andresuspended to approximately 2.0×10⁶ cells/ml in HBSS. Mice in the studygroups are injected with 0.10 ml of the cell suspension (s.c.) using a 1cc syringe fitted with a 25 g needle; injections are made on theanimal's left side, approximately mid-region. Tumors will develop inapproximately two weeks.

Study groups are assigned to separate study groups based on the creationof a comparable tumor size distribution in each group (average tumorsize, expressed as a product of length×width of the tumor, wasapproximately 80 mm²). 200 mg of the Fc engineered rituximab antibodiesis used for treatment. The groups are treated via tail-vein injectionsusing a 100 μl Hamilton syringe fitted with a 25 g needle.

Tumor measurements are made every two or three days using a caliper.

6.18 Clinical Studies of Fc Engineered Rituximab

Fifteen patients having histologically documented relapsed non-HodgkinsB cell lymphoma will be treated with Fc engineered rituximab in aclinical trial. Each patient will receive a single dose of antibody in adose-escalating study; there will be three patients per dose: 10 mg/m²;50 mg/m²; 100 mg/m²; 250 mg/m² and 500 mg/m². Treatment will be by i.v.infusion through an 0.22 micron in-line filter with the antibody beingdiluted in a final volume of 250 cc or a maximal concentration of 1mg/ml of normal saline. Initial rate will be 50 cc/hr for the firsthour; if no toxicity was seen, dose rate will be escalated to a maximumof 200 cc/hr.

Toxicity (as indicated by the clinician) will be judged on a range from“none”, to “fever” to “moderate” (two patients) to “severe” (onepatient). Peripheral Blood Lymphocytes will be analyzed to determine,inter alia, the impact of the antibodies on T-cells and B-cells.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed since these embodiments areintended as illustration of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Throughout this application various publications are cited. Theircontents are hereby incorporated by reference into the presentapplication in their entireties for all purposes.

1. A modified antibody that binds a CD32B antigen, said modifiedantibody comprising a variant human IgG1 Fc region, wherein said varianthuman IgG1 Fc region comprises at least one amino acid modificationrelative to the human IgG1 Fc region of a parent antibody that bindssaid antigen, said amino acid modification(s) comprising amino acidmodification(s) that alter the affinity or avidity of the variant Fcregion for binding to an FcγR such that said modified antibody istherapeutically effective in a patient refractory to treatment with saidparent antibody, said amino acid modification(s) that alter the affinityor avidity of the variant Fc region for binding to an FcγR consisting ofthe modification of 1, 2, 3, 4 or 5 amino acid residues of the IgG1 Fcregion of said parent antibody.
 2. The modified antibody of claim 1,wherein said modified antibody exhibits, in an in vitro assay,detectable effector function activity in cells derived from saidpatient, which cells are positive for said antigen, and said parentantibody does not exhibit detectable function activity in said cellsusing said in vitro assay.
 3. The modified antibody of claim 1, whereinsaid amino acid modification(s) that alter the affinity or avidity ofthe variant Fc region for binding to an FcγR comprise(s) a substitution:(i) at position 370 with glutamic acid, at position 396 with leucine andat position 270 with glutamic acid; (ii) at position 419 with histidine,at position 396 with leucine and at position 270 with glutamic acid;(iii) at position 240 with alanine, at position 396 with leucine and atposition 270 with glutamic acid; (iv) at position 255 with leucine, atposition 396 with leucine and position 270 with glutamic acid; (v) atposition 255 with leucine, at position 396 with leucine, at position 270with glutamic acid and at position 292 with glycine; or (vi) at position255 with leucine, at position 396 with leucine, at position 270 withglutamic acid and at position 300 with leucine.
 4. The modified antibodyof claim 1, wherein at least one of said amino acid modification(s) thatalter the affinity or avidity of the variant Fc region for binding to anFcγR is in a CH2 domain of said variant human IgG1 Fc region.
 5. Themodified antibody of claim 4, wherein said amino acid modification(s)that alter the affinity or avidity of the variant Fc region for bindingto an FcγR in said CH2 domain comprises a substitution at position 240,243, 247, 255, 270, 292, or 300 with another amino acid at thatposition.
 6. The modified antibody of claim 1, wherein at least one ofsaid amino acid modification(s) that alter the affinity or avidity ofthe variant Fc region for binding to an FcγR is in a CH3 domain of saidvariant human IgG1 Fc region.
 7. The modified antibody of claim 6,wherein said amino acid modification(s) that alter the affinity oravidity of the variant Fc region for binding to an FcγR in said CH3domain comprises a substitution at position 370, 392, 396, 419, or 421with another amino acid at that position.
 8. The modified antibody ofclaim 1, wherein said amino acid modification(s) that alter the affinityor avidity of the variant Fc region for binding to an FcγR comprises atleast one amino acid modification in the CH2 domain and at least oneamino acid modification in the CH3 domain of the Fc region.
 9. Themodified antibody of claim 1, wherein said amino acid modification(s)that alter the affinity or avidity of the variant Fc region for bindingto an FcγR is in the hinge region of the human IgG1 heavy chain.
 10. Themodified antibody of claim 8, comprising at least one amino acidmodification that alters the affinity or avidity of the variant Fcregion for binding to an FcγR in the hinge region of the human IgG1heavy chain.
 11. The modified antibody of claim 1 which variant IgG1 Fcregion specifically binds FcγRIIIA with a greater affinity than saidparent antibody binds FcγRIIIA.
 12. The modified antibody of claim 1which variant IgG1 Fc region specifically binds FcγRIIA with a greateraffinity than said parent antibody binds FcγRIIA.
 13. The modifiedantibody of claim 1 which variant IgG1 Fc region specifically bindsFcγRIIB with a lower affinity than said parent antibody binds FcγRIIB.14. The modified antibody of claim 11 which variant IgG1 Fc regionspecifically binds FcγRIIB with a lower affinity than said parentantibody binds FcγRIIB.
 15. The modified antibody of claim 12 whichvariant IgG1 Fc region specifically binds FcγRIIB with a lower affinitythan said parent antibody binds FcγRIIB.
 16. The modified antibody ofclaim 1 which detectably binds cells positive for said antigen, whichantigen is expressed at a density of 200 to 1,000 molecules/cell on saidcells.
 17. The modified antibody of claim 2 wherein said effectorfunction is antibody dependent cell-mediated cell cytotoxicity (ADCC).18. The modified antibody of claim 2 wherein said effector function isphagocytosis, opsonization, cell binding, rosetting, complementdependent cell mediated cytotoxicity (CDC), or antibody dependentcell-mediated cell cytotoxicity (ADCC).
 19. The modified antibody ofclaim 2 wherein said in vitro assay is performed using effector cellsand target cells, under conditions in which the effector cell:targetcell ratio is 10:1.
 20. The modified antibody of claim 1 which is amonoclonal antibody.
 21. The modified antibody of claim 1 which is achimeric, human or humanized antibody.
 22. The modified antibody ofclaim 1, in which the parent antibody has immunomodulatory activity. 23.A pharmaceutical composition comprising the modified antibody of claim 1and a pharmaceutically acceptable carrier.
 24. The modified antibody ofclaim 1, wherein said amino acid modification(s) that alter the affinityor avidity of the variant Fc region for binding to an FcγR consist ofthe modification of 1, 2 or 3 amino acid residues of the IgG1 Fc regionof said parent antibody.
 25. The modified antibody of claim 1, whereinsaid amino acid modification(s) that alter the affinity or avidity ofthe variant Fc region for binding to an FcγR consist of the modificationof 4 or 5 amino acid residues of the IgG1 Fc region of said parentantibody.
 26. The modified antibody of claim 2, wherein said in vitroassay is performed using effector cells and target cells, underconditions in which the effector cell:target cell ratio is 30:1.
 27. Themodified antibody of claim 2 wherein said in vitro assay is performedusing effector cells and target cells, under conditions in which theeffector cell:target cell ratio is 50:1.
 28. The modified antibody ofclaim 2 wherein said in vitro assay is performed using effector cellsand target cells, under conditions in which the effector cell:targetcell ratio is 75:1.
 29. The modified antibody of claim 2 wherein said invitro assay is performed using effector cells and target cells, underconditions in which the effector cell:target cell ratio is 100:1. 30.The modified antibody of claim 1, wherein said modified antibodyexhibits, in an in vitro assay, detectable cell killing in cellspositive for said antigen, and said parent antibody does not exhibitdetectable cell killing in said cells using said in vitro assay.
 31. Themodified antibody of claim 30 wherein said in vitro assay is performedusing effector cells and target cells, under conditions in which theeffector cell:target cell ratio is 10:1.
 32. The modified antibody ofclaim 30 wherein said in vitro assay is performed using effector cellsand target cells, under conditions in which the effector cell:targetcell ratio is 30:1.
 33. The modified antibody of claim 30 wherein saidin vitro assay is performed using effector cells and target cells, underconditions in which the effector cell:target cell ratio is 50:1.
 34. Themodified antibody of claim 30 wherein said in vitro assay is performedusing effector cells and target cells, under conditions in which theeffector cell:target cell ratio is 75:1.
 35. The modified antibody ofclaim 30 wherein said in vitro assay is performed using effector cellsand target cells, under conditions in which the effector cell:targetcell ratio is 100:1.